ISSN 1061-9348, Journal of Analytical Chemistry, 2009, Vol. 64, No. 7, pp. 657673. Pleiades Publishing, Ltd., 2009.

. Original Russian Text S.V. Khlyntseva, Ya.R. Bazel, A.B. Vishnikin, V. Andruch, 2009, published in Zhurnal Analiticheskoi Khimii, 2009, Vol. 64, No. 7, pp. 677693.

REVIEWS

Methods for the Determination of Adenosine Triphosphate and Other Adenine Nucleotides
S. V. Khlyntsevaa, Ya. R. Bazelb, c, A. B. Vishnikina, and V. Andruchb
aDnepropetrovsk

State University, pr. Gagarina 72, Dnepropetrovsk, 49040 Ukraine University, Moyzesova 11, Kosice, 04001 Slovakia cUzhgorod State University, ul. Pidgirna 46, Uzhgorod, 88000 Ukraine
bSafarik

Received February 29, 2008; in nal form, October 20, 2008

AbstractThe following methods for the determination of adenosine triphosphate reported in the past 25 years are considered: bioluminescence methods with the use of the rey luciferase enzyme (with sensitivity to 1014 M); chromatographic methods (ion-exchange, thin-layer, and high performance liquid chromatography) for the determination of adenine nucleotides in mixtures with other nucleotides, nucleosides, and nitrogen bases; and uorescence, spectrophotometric, and electrochemical techniques (including those with the use of sensors), which are promising but not commonly used for the determination of adenine nucleotides. The advantages and disadvantages of these methods are demonstrated. DOI: 10.1134/S1061934809070028

Adenosine triphosphate (ATP) is a natural constituent of human and animal body tissues. It is formed in oxidation reactions and in the course of the glycolytic cleavage of carbohydrates. ATP is a starting substance in the synthesis of nucleic acids; it participates in the regulation of many biochemical processes and is a mediator in synapses. ATP also participates in metabolic processes. It interacts with actomyosin to decompose into adenosine diphosphate (ADP) and inorganic phosphate. In this case, energy is released, and the major portion of this energy is used by muscles to perform mechanical work and to synthesize protein, urea, and metabolic intermediates. Thus, the main function of ATP in the body is to supply energy for many biochemical reactions. ADP is an important regulator of many in vitro metabolic processes, including oxidative phosphorylation, glycolysis, glycogenesis, and ion transport [1]. It is well known [1, 2] that the energy level of a cell depends on the balance between adenosine phosphates (ATP, ADP, and adenosine monophosphate (AMP)). Atkinson and Walton [3] proposed so-called energy charge (EC), which is the mole fraction of adenine acid charged by its conversion into ATP, as a fundamental parameter of metabolic control: [ ATP ] + 1/2 [ ADP ] -. EC = ---------------------------------------------------------------[ ATP ] + [ ADP ] + [ AMP ] The above value can vary from zero (in the presence of only AMP) to 1.0 (which suggests the complete conversion of AMP molecules into ATP). In this case, ADP is considered as a semi-charged species. Two important conclusions can be made on this basis. When the EC is

higher than 0.5, the system of the use of ATP increases its efciency. However, when the EC decreases to 0.5, ATP regeneration is predominant in the system. Thus, for example, it was found that the EC in the cells of growing bacteria was maintained at a level of 0.8, and the value of EC was close to 0.5 in aged cells. At even lower values of EC, the cells lost their activity [2]. This approach allows one to use ATP as an indicator of cell viability and damage. Moreover, the amounts of bacteria, living biomass, and microbiological activity can be judged from the concentrations of ATP in environmental samples. Therefore, the concentrations of ATP are frequently used for health control. However, in some cases, the determination of only ATP is insufcient and all of the adenine nucleotides should be determined. In particular, this simultaneous determination of ATP, ADP, and AMP is very important for product quality control and in the course of process monitoring and clinical judgment of ATP-containing pharmaceuticals [2, 4]. BIOLUMINESCENCE METHODS FOR THE DETERMINATION OF ADENINE NUCLEOTIDES Bioluminescence methods with the use of the rey luciferase enzyme are most commonly used for the determination of adenine nucleotides. The advantages of these methods are their high sensitivity, selectivity (with the use of puried enzymes), and relative ease of application. The reaction based on luciferinluciferase was discovered as early as 1884 [5]. However, it is evident that

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even milliseconds). For this purpose, the following two procedures were proposed: (1) rapid cell lysis and almost instantaneous determination of ATP; (2) ATP extraction (to deactivate enzymes that hydrolyze ATP) [11]. In the latter case, the use of the following extractants was recommended: (1) buffer solutions (for example, a Trisborate buffer with pH 9.2) [13, 14]; (2) dilute solutions of acids (perchloric and trichloroacetic acids) with subsequent acid neutralization [13, 1517]; (3) surfactants (for example, bezalconium chloride and Triton X-100) [15, 17, 18]; (4) organic solvents [17, 19]. A particular extractant of choice depends on the type of cells. However, all of the extractants adversely affect the reaction of ATP with luciferinluciferase to decrease the activity of luciferase and, correspondingly, to decrease the sensitivity of the method. Yang et al. [13] proposed the use of boiling deionized water, which has almost no effect on bioluminescence but efciently inhibits ATPase, for the extraction of cellular ATP. The use of diethylaminoethyldextran, which enhances the bioluminescence reaction in the presence of the mentioned Triton X-100 and trichloroacetic acid, was proposed [15, 20, 21] to enhance bioluminescence emission. Kamidate et al. [18] proposed the use of liposomes containing phosphatidylcholine and cholesterol for this purpose; these liposomes can bind bezalconium chloride to form cationic liposomes. However, the main problem in the determination of intracellular ATP is the interference of extracellular ATP, whose concentration can be higher by several orders of magnitude. To eliminate this interference, dephosphorylating enzymes, such as apyrase [22, 23] and ATPase [22], or enzyme mixtures were used. For example, Sakakibara et al. [24] used adenosine phosphate deaminase and apyrase for the conversion of extracellular ATP and similar adenosine derivatives into inosine monophosphate (IMP), which is inactive in the luciferinluciferase system. Another serious problem in this method is its low selectivity with respect to many inorganic ions, which interfere with the determination to decrease considerably the emission of the luciferinluciferase system. The principle of this interference consists in the ability to be coextracted with ATP or to be a part of the extraction system (e.g., in extraction with buffer solutions). The interference of various cations and anions has been studied (see, for example, [2530]). A decrease in the
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Fig. 1. Structural formulas of (a) D-luciferin and (b) oxyluciferin.

detailed studies of rey luciferase started in 1947 when McElroy [6] was the rst to apply this reaction to the determination of ATP. The method was based on the measurement of time to the disappearance of emission; even under conditions of visual process control, this method made it possible to determine ATP contents in the range of 10150 g [7]. Currently, the mechanism of this reaction has been studied in sufcient detail. It is based on the oxidation of D-luciferin (Fig. 1) in the presence of ATP and oxygen catalyzed by rey luciferase: ATP + D-luciferin + O2 luciferase, Mg AMP + oxyluciferin + PP + O2 + h, where PP is pyrophosphate. The emission spectrum in the region of 470700 nm is asymmetric with a maximum at 562 nm. The quantum yield is 0.9 einstein/mol luciferin, and the emission intensity is proportional to the concentration of ATP. It is of interest that the enzyme activity was lost upon the fractionation of rey extracts with ammonium sulfate; however, it was readily restored upon the addition of metal ions. An increase in the activity was noted in the following order of metals: Zn < Ni < Fe < Co < Mn < Mg [8]. More than 100 bioluminescence procedures developed before 1981 for the determination of ATP with the use of rey luciferase were described [2, 7]. For the most sensitive of these procedures, the detection limit of ATP was at the fmol level [9, 10]. However, interest in this method remains high: procedures that are even more sensitive are under development with the use of new instrumentation, automation, and computer technologies. Because of its high sensitivity, the bioluminescence method can be used for the determination of intracellular ATP [11, 12]. However, because of cell degradation, an additional stage is required for the stabilization of the cell state over a very short time interval (seconds or
2+

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interference of cations was found in the following order: Cu2+ > Zn2+ > 2+ > + > Na+ > Rb+ > Li+. Other studies [28, 30] suggest the interference of other ions: Mg2+, Mn2+, Ti3+. Hg2+, and Cr3+. The interference of cations can be decreased with the use of EDTA [28]. This kind of order for anions is as follows: O 3 > O 4 > S O 4 > I ~ N O 3 > Br > r > F > 3. The bioluminescence reaction occurs very rapidly (2030 s) and begins almost instantaneously after the addition of reagents when the test sample and the reagents are incompletely mixed. In this time interval, the emission intensity can depend on not only the concentration of ATP but also random factors (such as the intensity of stirring). Several mathematical approaches were proposed to increase the sensitivity of this method by excluding the reagent stirring time from the total reaction time [31, 32]. Among them are robust exponential regression and outlier detection (RER) [31, 32], the SavitzkyGolay smoothing method (SSG), and the Kalman Filter smoothing method (SKF) [31]. The use of these approaches considerably decreases the relative standard deviation (RSD) (from 25 to 7%) and the limit of detection (LOD) (from 6 109 to 6.6 1010 M ATP). Based on enzyme reactions, an amplication procedure for the determination of ATP [33] with the use of adenylate kinase (ADK) and polyphosphate kinase (PPK) was developed. This approach is based on the following reaction scheme:
AMP + ATP 2ADP + polyPn 2AMP + 2 ATP 4ADP + polyPn 2
ADK PPK ADK PPK 2 3 2

instrument used for luminescence intensity measurements. Several procedures with the use of the bioluminescence determination of ATP in ow injection analysis were determined [35, 36]. This system [35] involves the use of a double injection pump, which simultaneously injected discrete portions of a sample containing ATP and enzyme solutions. With a corresponding conguration of the injection pump, any portion of unused enzyme solution is recycled to save this very expensive reagent. Other adenine nucleotides are rarely determined. Tanaka et al. [37] proposed the use of phosphotransferase and inorganic polyphosphates for the conversion of AMP into ADP. This method does not require ATF for initiating the reaction; therefore, it exhibits lower background luminescence. In the majority of cases, ADK and pyruvate kinase (PK) are used in the determination of ADP and AMP for converting them into ATP followed by the determination of its concentration by the standard bioluminescence method [19]: AMP + CTP
ADK

ADP + CDP;

2ADP 2 ATP + polyPn 2 4ADP 4 ATP + polyPn 6

After amplication, ATP is determined by the standard bioluminescence method using the reaction with rey luciferase. This procedure increased the sensitivity of the determination of ATP by a factor of 10000, as compared with that of the traditional bioluminescence method. Based on an amplication system of this type, two mathematical models for the determination on ATP were developed [34]. In this case, the concentration of ATP was monitored based on the time taken to reach a luminescence maximum (instead of the direct luminescence measurement). Consequently, the sensitivity of the method was not restricted by the capabilities of the
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ADP + phosphoenolpyruvate PK ATP + pyruvate. Sakakibara et al. [38] described a highly sensitive procedure for the determination of ATP and AMP; this procedure is based on the use of pyruvate phosphate dikinase (PPDK). Sensitivity was increased by ATP regeneration from AMP and pyrophosphate, which were obtained in the reaction with luciferinluciferase. The background luminescence of the reagent was decreased with the use of adenosine phosphate deaminase, and an excess of this latter substance was bound by coformycin, which is an inhibitor of adenosine phosphate deaminase and prevents the decomposition of ATP in the sample. Schultz et al. [39] implemented a similar approach for the determination of ADP in the presence of a large amount of ATP. In this case, the effect of endogenous ATP was removed by the hydrolysis of ATP upon the addition of the ATP sulfurylase enzyme. An excess of this enzyme was inactivated by heating, whereas ADP was converted into ATP with PK. Table 1 gives comparative characteristics of various bioluminescence methods for the determination of adenine nucleotides (most frequently, ATP). CHROMATOGRAPHIC METHODS Chromatographic methods are commonly used for the simultaneous determination of nucleotides in a mixture. Brown with coauthors [50, 51] surveyed methods for the determination of nucleotides, nucleosides, and nitrogen bases, which were known before 1981. The ana2009

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Table 1. Bioluminescence methods for the determination of adenine nucleotides Procedure 5 1095 105 (5025000 cells) ATP (17 10) 1015 mol of ATP Extracts from yeast and beef, beer, milk, and rice Extracts from Escherichia coli Water (tap, drinking, sterilized, and natural water) Juice, beer, condensed milk, yeast extract, fish extract, and malt extract 4 10134 109 ATP and 4 1013 ATP and AMP AMP 1018 mol of ATP Analytical range, M cmin (detection limit), M Test material Reference [11] [40] [38]

Determination of ATP after rapid cell lysis

Determination of ATP, creatine phosphate, and creatine followed by the calculation of ADP and AMP concentrations

Cyclic method with the use of PPDK (for the regeneration of ATP and adenosine phosphate deaminase and a decrease in the background luminescence of the reagent

Amplification procedure based on (1) adenylate kinase for the conversion of ATP + AMP into 2ADP; (2) polyphosphate kinase for the conversion of ADP into ATP 107104 AMP

[33]

Procedure for the determination of AMP based on (1) the conversion of AMP into ADP with the use of phosphotransferase and inorganic polyphosphates; (2) the conversion of two molecules of ADP into ATP + AMP with the use of adenylate kinase

[37]

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Determination of AMP, ADP, and ATP based on (1) the conversion of AMP into ADP with the use of adenylate kinase; (2) the conversion of ADP into ATP with the use of pyruvate kinase 103106 cells ATP 5 1091 104 (800100000 cells) ATP

1.5 1012 mol in an ADP sample 15 1012 mol in an AMP sample 3.0 1013 ATP (in the presence of TCA) 5.0 1013 ATP (in the presence of Triton X-100)

Extracts from Escherichia coli

[19]

The use of boiling water for the extraction of cellular ATP

Extracts from Escherichia coli

[13] [11] [15]

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Extraction of ATP with perchloric acid (for the deactivation of enzymes that hydrolyze ATP)

The use of diethylaminoethyldextran to enhance bioluminescence 3.0 10133.0 109 ATP emission in the presence of the extractants of ATP (Triton X-100 (in the presence of TCA) and trichloroacetic acid (TCA)) 5.0 10133.0 109 ATP (in the presence of Triton X-100) 4 1034 106 cell/mL ATP

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Determination of ATP in the presence of the cationic surfactant bezalconium chloride (for the extraction of ATP from cells) and liposomes containing phosphatidylcholine and cholesterol (for binding an excess of bezalconium chloride)

25 1015 mol of ATP

Extracts from Escherichia coli

[18]

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Automated method based on regression analysis for excluding the time of mixing the reagents from the total reaction time

8 1010 ATP

Marine bottom sediments

[32]

Table 1. (Contd.) Procedure Fish extract; Analytical range, M cmin (detection limit), M Test material Reference [41]

Bioluminescence method for the determination of ATP improved 1 1065 105 ATP by increasing the enzyme concentration in the presence of bovine serum albumin, dithiothreitol, and EDTA 1 10131 1012 mol of ATP, ADP, and AMP Mouse embryo

The use of purified enzymes for the determination of ATP (luciferase) and for the conversion of ADP and AMP into ATP (adenylate kinase) 1 1012 ATP 20 cells 1 1011 ATP

[12]

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The use of firefly luciferase retained by silica gel modified with sugars (using a solgel synthesis) 1 10113 106 ATP

[42]

The use of firefly luciferase covalently immobilized on collagen

Human blood

[43] [44]

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Fiber optics sensor based on three enzymes (adenylate kinase, creatine kinase, and firefly luciferase)

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2.5 10122.5 109 mol 2.5 1012 mol of ATP, of ATP, 1 1011 mol of ADP, 11 9 1 10 2.5 10 mol 2.5 1011 mol of AMP of ADP, 2.5 10115 109 mol of AMP 2.5 1013 mol of ATP

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Bioluminescence fiber optics sensor for the determination of ATP 1 10101 106 ATP based on firefly luciferase coimmobilized with a bacterial luminescent system on a polyamide membrane

[45, 46]

Biosensor based on coimmobilized luciferase and poly-L-lysine on nonporous glass

5 108 ATP

[47]

Flow injection analysis

1.0 10104.0 108 ATP 1010 ATP 3 10131 1010 mol of ATP 1 10125 1010 mol of ATP 1014 ATP 3 1013 mol of ATP

Extracts from bacteria

[35] [36] [48]

Flow analysis with the use of firefly luciferase immobilized on a nylon matrix

Flow analysis with the use of firefly luciferase immobilized on a methyl methacrylate support

3 1013 mol of ATP

Extract from thrombocytes

[49] 661

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lytes were separated by ion-exchange chromatography [50, 5256] and reversed-phase [50, 5767] or ion-pair reversed-phase [63, 6882] high-performance liquid chromatography (HPLC). Thin-layer chromatography was also used for the determination of ATP [83, 84]. The disadvantage of ion-exchange chromatography is the low stability of ion-exchange columns; because of this, separation is not always adequately reproducible [51]. In the case of reversed-phase HPLC, gradient elution with a buffer solution should be used; this results in a considerable drift of the baseline. Moreover, the procedure required for the equilibration of the column after use is vary complicated. The disadvantages of the ion-pair version of HPLC are the short service life of columns and the complicated procedure of quantitative analysis [62]. The measurement of light absorption in the UV region at 210 [53, 58] or 254 nm [57, 61, 62, 65, 66, 82, 8587] and the measurement of uorescence emission intensity [64, 67, 68, 70, 72, 77, 84] are commonly used for detection. In the latter case, the reaction of adenine-containing compounds with halogen acetaldehydes is used. In this case, the following two versions are possible: (1) the reaction with chloro- or bromoacetaldehyde is performed before passing the mixture through a column and the 1,N6-ethene derivatives of ATP, ADP, AMP, and other adenine-containing compounds are separated by chromatography [64, 68, 70, 72]; (2) a uorescent product is obtained after the separation of nucleotides on a column [77]. The reaction with chloroacetaldehyde is slow; therefore, it is performed at a high temperature for acceleration (30 min at 100). Bromoacetaldehyde is more reactive (8 min at 100) but less accessible. A greater problem is that nucleotides can undergo hydrolysis at an elevated temperature even in a weakly acidic medium to result in a decrease in the concentration of ATP and in an increase in the concentrations of ADP and AMP [77]. Hu et al. [76] proposed to use the reaction of phosphates formed upon the hydrolysis of ATP with molybdate and a reducing agent in an acidic medium with the formation of a heteropoly complex (the molybdophosphoric blue method) for the determination. Light absorption was measured at 880 nm. Isocratic or gradient elution can be performed with the use of reversed-phase HPLC for the determination of nucleotides. In the former case, a phosphate or citrate buffer solution is used [62, 71, 72, 87]. Gradient elution is performed by adding acetonitrile [69, 77, 78, 85, 86], methanol [60, 61, 63, 66, 82], or their mixture [58] to the test solution. At the same time, Ozogul et al. [86] found that acetonitrile or methanol did not improve separation but only shortened the analysis time. Isocratic elution can also be performed with a mixture of a

phosphate buffer solution and acetonitrile [57, 64, 68, 69, 74] or methanol [70, 73]. Tetrabutylammonium is the most effective counterion in the ion-pair version of reversed-phase HPLC [79]; triethylamine can also be used [71]. The advantage of chromatographic techniques is the possibility of performing the separation and simultaneous rapid determination of not only nucleotides but also nucleosides, nitrogen bases, creatine, and creatine phosphate [53, 57, 58, 78]. Thus, for example, one of the most rapid procedures [58] made it possible to separate creatine, creatine phosphate, ATP, ADP, AMP, adenosine, inosine, hypoxanthine, xanthine, and nicotinamide adenine dinucleotide (NAD+) for 5 min with the use of gradient elution with a mixture of acetonitrile and methanol. Ryll and Wagner [81] separated 30 nucleotides in 25 min using methanolpH gradient elution. Table 2 compares the characteristics of well-known chromatographic methods for the determination of various nucleotides. FLUORESCENCE METHODS Fluorescence can be used not only as a detection technique in the chromatographic determination of nucleotides but also as an independent analytical technique. The well-known uorescence methods for the determination of ATP can be subdivided into the following two groups: (1) determination based on uorescence enhancement; (2) determination based on uorescence quenching. The former group of methods is based on the uorescence enhancement of the complexes of rare earth elements (Eu and Tb) with doxycycline [88], noroxacin [89], oxytetracycline [90], and phenanthroline [91] in the presence of ATP. Li et al. [92] proposed a more complex system (TbGdATPphenanthroline), in which the uorescence of the TbATPphenanthroline complex was decreased byGd3+. Foy and Pacey [93] proposed the use of the effect of uorescence enhancement upon the formation of a chelate of ATP with N-(anthracen-9'-yl methyl)tris(3-aminopropyl)-amine. However, ADP also gave an analytical signal. The applicability of this approach to the determination by ow injection analysis was demonstrated. Pivovarenko et al. [94] developed a uorescence method for the determination of ATP based on the appearance of a new band in the uorescence spectrum of 3-hydroxy-4'-(dimethylamino)avone (FME) in the presence of ATP. They explained this phenomenon by the formation of an FMEATP associate, in which the FME molecule is stabilized by the tetracharged ATP anion. Li et al. [95] proposed the use of a reaction between a polythiophene derivative and ATP, as a result of which
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Table 2. Chromatographic methods for the determination of adenine nucleotides Analytes AMP, ADP, and ATP AMP, NAD+, IMP, GMP, NADP+, ADP, GDP, 20400 pmol CTP, UTP, ATP, GTP, creatine, and creatine (20 pmol of AMP) phosphate Normal and anoxic rat heart AMP, ADP, ATP, GTP, GDP, IMP, and NAD+ 2.0 mmol/kg ATP 0.5 ng of ATP Soils Erythrocyte extracts Human thrombocytes Human erythrocytes Lychee fruits and pericarp tissue Porcine myocardial tissue Herring Rat liver Mammalian skeletal muscles AMP, ADP, and ATP ATP, IMP, creatine, and creatine phosphate ATP, ADP, ATP, IMP, inosine, and hypoxanthine ATP AMP, ADP, ATP, NADP+, and NAD+ AMP, ADP, ATP, and AMP, ADP, ATP, NADP+, and NAD+ AMP, ADP, and ATP NAD+ Human erythrocytes Calibration graph linearity range (cmin) Test material Reference [52] [53]

Type of chromatography

Detection technique

Ion-exchange chromatography

UV detector 210 nm

[55] [62] [57] [86] [87] [61] [65] [66] [85] [58]

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Reversed-phase HPLC

UV detector 254 nm

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UV detector 210 nm

Creatine, creatine phosphate, ATP, ADP, AMP, 0.2500 mol/L adenosine, inosine, hypoxanthine, xanthine, and (0.2 mol/L) ATP, NAD+ ADP, and AMP AMP, ADP, ATP, IMP, inosine, and hypoxanthine ATP, ADP, AMP, and adenosine 2200 g/g ATP, ADP, and AMP

UV detector

Fresh and canned fish 0.120 pmol of ATP, Pulmonary artery ADP, and AMP (0.1 pmol) Rat liver mitochondria Human or rat muscle tissue Extracts from cardiac tissue, muscles, brain, liver, erythrocytes, and yeast cells

[63] [64]

Fluorescence

3'-AMP AMP, ADP, ATP, IMP, inosine, hypoxanthine, xanthine, and uric acid Nucleotides, nucleosides, and purine bases

[67] [60] [59]

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Table 2. (Contd.) Analytes Adenine, adenosine, AMP, ADP, and ATP ATP, ADP, and AMP AMP, ADP, ATP, and adenosine AMP, ADP, and ATP ATP, ADP, and AMP AMP, ADP, ATP, GTP, GDP, IMP, NADP+, NAD+, ADP-ribose; inosine, adenosine, hypoxanthine, and xanthine AMP, ADP, ATP, IMP, inosine, and hypoxanthine ATP AMP, ADP, and ATP AMP, ADP, and ATP IMP, AMP, ADP, ATP, and cAMP Nucleotides and nucleotide sugars Creatine, creatine phosphate, AMP, ADP, ATP, GTP, GDP, IMP, NAD+, inosine, adenosine, hypoxanthine, and xanthine AMP, ADP, and ATP 106 M 40 pmol 20 pmol 0.1100 nmol/mL (10 pmol) 54000 pmol (25 pmol) Rat kidney Extract from mouse liver Integument of the mussel Mytilus galloprovncialis Lmk Human erythrocytes 0.2 ATP, 0.1 ADP, and 0.05 AMP, g/g Soil 0.110.0 pmol Rat artery (ATP, ADP, and AMP) Calibration graph linearity range (cmin) Test material Reference [68] [70] [72] [77] [69] [82]

Type of chromatography

Detection technique

Ion-pair reversed-phase chromatography

Fluorescence

UV detector in the region of 220320 nm

UV detector 254 nm

UV detector

2200 g/g ATP, ADP, Fresh and canned fish and AMP 1733 mol/L Fish Rat brain extract Biological extracts, including a cardiac muscle extract Extracts from fibroblasts and erythrocytes Extracts from cardiac tissue Porcine and rat cardiac muscles

[63] [76] [71] [73] [74] [75] [80] [78]

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Thin-layer chromatography

Fluorescence

Escherichia coli cells Rat liver

[83] [84]

2009

Fluorescence

METHODS FOR THE DETERMINATION OF ADENOSINE TRIPHOSPHATE Table 3. Fluorescence methods for the determination of ATP Complex Eudoxycycline Tbnorfloxacin TbGdphenanthroline Euoxytetracycline Tbphenanthroline Euenoxacinphenanthroline Ruphenanthrolinedipyrido[3,2-a:2',3'-c]phenazine Zndipicolylamine N-(Anthracen-9'-yl-methyl)tris(3-aminopropyl)amine 3-Hydroxy-4'-(dimethylamino)flavone Chloroacetaldehyde Anthrylmethylamine a/f, nm 385/612 335/545 300/545 /612 340/616 /460 335/417 /415 Calibration graph linearity range, M 1.00 1072 106 1.00 1061.6 105 1 1075 105 106 8.00 1081.50 cmin, M 4.07 108 4.13 108 5.4 109 109 Tablets Tablets Injections 106 200 pmol Thrombocytes and blood plasma _ 0.034 g/mL 108 2.67 Test material Injections and tablets Injections and tablets Tablets

665

Reference [88] [89] [92] [90] [96] [91] [97] [99] [100] [93] [94] [101] [102]

Euibuprofenphenanthroline 273/615

0.110 g/mL 1.0 1061.0 105 0.410 g/mL 0.5100 ppm 103105

0.03 g/mL

the color of the solution changed from yellow to pink. The possibility of spectrophotometric determination was considered in addition to uorescence determination. The well-known ability of ATP to quench the uorescence of rare earth metal complexes formed the basis of a few procedures for the determination of ATP [96, 97]. Thus, Zhao et al. [98] studied in detail the uorescence quenching mechanism of the b3+ complex with tiron in the presence of nucleotides, polynucleotides, and nucleic acids. They found that the uorescence quenching effect was due to the presence of a phosphate moiety in the test molecules; this moiety competes with tiron in the course of binding to b3+ to form a nonuorescent binary complex under optimum conditions. Table 3 summarizes the comparative characteristics of uorescence techniques for the determination of ATP. SENSORS Sensors are convenient tools for the determination of ATP; the majority of them were developed with the use of immobilized enzymes. The use of an 22-sensitive electrode with immobilized glucosidase and hexokinase for the measurement of ATP concentrations was proposed [103, 104]. The method is based on the following reactions: glucose + O2 gluconic acid + 2O2;
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glucose + ATP

glucose-6-phosphate + ADP.

A catalyst (glucose oxidase) is required for the former reaction to occur, whereas hexokinase and Mg2+ ions as a cofactor are necessary for the latter reaction. The amount of the resulting hydrogen peroxide decreases in the presence of ATP; this makes it possible to measure the concentration of ATP. Scheller with colleagues proposed the use of an oxygen-sensitive electrode with immobilized hexokinase and glucose oxidase [105] or glucose-6-phosphate [106]. Adachi et al. [107] developed a potentiometric ATP sensor with a detection limit of 1.0 M based on a lipid bilayer membrane with Na+,K+-ATPase. Bcking et al. [108] immobilized ATPhase (F0F1-+-ATPhase) on a gold electrode. Thus, they obtained a system of two ATPhase complexes: one of them (F0) occurred at the center of the membrane, and the other (F1) occurred on its surface. ATPhase transferred protons through the membrane and hydrolyzed ATP to ADP. Sensors in which immobilized rey luciferase was used have been described in a number of publications (see Table 1). The rst attempts to immobilize luciferase on various materials were made as early as the late 1970s [7]. A polyamide membrane [45, 46], glass [47], nylon [48], methacrylate [49], lter paper [109], collagen [43], etc., were used for immobilization. More recently, multifunctional ber optics sensors
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for the simultaneous determination of ATP and NAD+ were developed [45, 46]. Michel et al. [44] proposed a sensor for the determination of ATP, ADP, and AMP with the use of two enzymes (adenylate kinase and creatine kinase) in addition to the above luciferase. These enzymes were coimmobilized on a collagen membrane, whereas luciferase was bound to a separate membrane. Because of the inhibiting effect of ATP on the activity of adenylate kinase, this sensor made it possible to measure ATP concentrations over the range of 2.52500 pmol even in the samples containing all of the three nucleotides. Several uorescence sensors have been developed for the determination of ATP [100, 102, 110112]. Ojida et al. [100] developed a sensor based on the dipicolylaminezinc complex, which coordinatively binds to ATP and provides an opportunity to determine ATP concentrations in neutral aqueous solutions. Descalzo et al. [102] developed a uorescence chemosensor with immobilized anthrylmethylamine groups. In the presence of trace amounts of ATP, uorescence quenching at 415 nm was observed. Marcotte and Taglietti [110] proposed another principle for the development of a uorescence chemosensor. The uorescent indicator coordinatively binds to the corresponding receptor (polyazamacrocyclic complex of copper [u2(L)]) and loses the ability to emit light. At the same time, upon substrate binding to the receptor, the indicator passes into solution with the compete restoration of uorescence emission. The indicator of choice made it possible to develop a sensor for the determination of ATP concentrations at a micromolar level in physiological solutions. Several optical sensors were developed for the determination of ATP based on tetrabrucineporphyrin [113] and 1,3,5-triarylpent-2-en-1,5-diones [114], which made it possible to determine the concentration of ATP in the presence of ADP and AMP. In 1988, Umezawa et al. [115] were the rst to use a macrocyclic polyamine as the active membrane component of an ion-selective electrode and to develop a potentiometric sensor for the determination of ATP with a detection limit of 0.1 M on this basis. Radecka and coauthors [116, 117] continued the development of sensors based on macrocyclic polyamines for the determination of ATP, ADP, and AMP. Srinivasan et al. [118] proposed two new sensors based on ribonucleic acid for the determination of ADP in the presence of a 100-fold amount of ATP. A potentiometric method for the determination of ATP (cmin = 109 M) with the use of a choline-sensitive electrode should also be noted [119]. Choline quantitatively reacts with ATP to form choline phosphate, to which the electrode is insensitive. Because of this, the

concentration of ATP corresponds to the amount of reacted choline. OTHER METHODS Among other methods, we note an original spectrophotometric method for the determination of ATP [120] based on the well-known reaction of orthophosphate with molybdenum to form molybdophosphoric blue. Apyrase (from potatoes) was used for the hydrolysis of ATP; it catalyzes the hydrolysis of ATP to AMP and two orthophosphate ions. In this method, the detection limit of ATP was 0.15 M, Han et al. [121] demonstrated the spectrophotometric determination of AMP, which substituted for pyrocatechol violet in the corresponding complex of zinc. In this case, the decrease in the absorption band intensity of the complex at 620 nm was proportional to the concentration of AMP over the range of 1050 M. Perez-Ruiz et al. [122] described the determination of ATP with the use of the photochemical and chemiluminescence reactions of glucose with ATP in a ow injection system in the presence of catalysts (hexakinase and Mg2+). An excess of glucose was oxidized by 9,10-anthraquinone-2,6-disulfonate with the release of hydrogen peroxide, which was determined using a chemiluminescence reaction with luminol. In this method, the determination limit of ATP was 0.07 mg/L. Hansen et al. [123] described another ow injection system. In this case, the determination was performed using the uorescence of the resulting NAD+. The determination limit was 15 nM. Several amplication procedures, including ow injection analysis, were developed for the determination of ATP and ADP with the use of cyclic reactions with the participation of enzymes [123127]. In the course of ow injection analysis with amperometric detection [124], ATP was determined in the system with an enzyme reactor including coimmobilized pyruvate kinase and hexokinase in the presence of glucose, NAD+, and phosphoenolpyruvate. In this case, the resulting glucose-6-phosphate was converted into an equivalent amount of NAD+. This latter was determined with the use of a modied graphite electrode. The detection limit of ATP was 1.0 nM. Amperometric detection in combination with capillary zone electrophoresis [4, 128] was used for the simultaneous determination of ATP, ADP, and AMP. Lin et al. [129] proposed a similar principle for the determination of AMP (up to 9.0 1012 M) in mixtures with purine bases and ribonucleosides. Ikeda et al. [130] developed an original procedure for the immobilization of alkaline phosphatase on glass. It is well known that the hydrolysis of adenosine phosphates under the action of alkaline phosphatase results in the formation of alcohol and phosphate ions.
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METHODS FOR THE DETERMINATION OF ADENOSINE TRIPHOSPHATE Number of publications 14 Bioluminescence 12 10 8 6 4 2 0 Fluorescence

667

Chromatography Sensors

19831987 19881992 19931997 19982002 20032007 Years

Fig. 2. Diagram of the development of methods for the determination of adenine nucleotides.

The concentrations of ATP, ADP, and AMP were found from the concentration of phosphate ions determined by conductometry. Lactic acid; creatine; myoglobin; albumin; and chloride, nitrate, phosphate, and sulfate ions in a ratio of 1 : 1 caused no interference with the determination; however, other phosphorus-containing compounds, which can also react with alkaline phosphatase, interfered. The method is applicable to the monitoring of ATP (0.010.1 mM), ADP (0.050.2 mM), and AMP (0.010.1 mM) in individual samples and to the determination of the total concentrations of these analytes. BenBashat et al. [131] determined the concentration of ADP in the presence of ATP using 31P NMR spectroscopy. Miot et al. [132] proposed the use of -ray emission from 31P nuclei for the determination of ATP. CONCLUSIONS In conclusion, note that a great many methods have been developed for the determination of adenine nucleotides (primarily, ATP). First, this can be explained by the paramount biological importance of these substances. Figure 2 illustrates the dynamics of publications on the most important methods for the determination of adenine nucleotides over the past 25 years. At the same time, interest in the development of new and more efcient methods for the determination of adenine nucleotides has not decreased and it is too early to suggest an ideal method. Each of the described methods has its advantages and disadvantages. Bioluminescence methods are the most sensitive (the limit of detection can be as low as 1014 M) and they are most
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widely used in the analysis of real samples. These methods are sufciently sensitive even for the determination of intercellular ATP. Therefore, they are primarily used for the detection of microbiological contaminations and the determination of the numbers of bacteria in the food industry, clinical diagnostics, etc. (see Table 4). The capabilities of the bioluminescence technique for the determination of ATP have been compared with those of standard microbiological methods for the determination of bacteria (CFU methods) [136, 143, 157, 190]. It was noted that the results of the determination of bacteria from the concentration of ATP correlated well with the results obtained using standard methods. However, the bioluminescence technique is much faster because it does not require the bacterial growth procedure. Hawronskyj and Holah [191] described in detail the advantages and disadvantages of various methods for the determination of ATP as applied to food health monitoring. However, the bioluminescence technique remains expensive because of the use of luciferinluciferase; thus, it is unsuitable for routine analyses. Chromatographic techniques are less sensitive but more selective, primarily, because of combining separation and determination stages. They make it possible to determine adenine nucleotides in complex mixtures with other nucleotides, nucleosides, and nitrogen bases. Other techniques, mainly those related to the use of sensors, are promising. However, these techniques are not widely used, and they cannot currently compete with bioluminescence and chromatographic techniques for the determination of adenine nucleotides.
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Table 4. Use of methods for the determination of adenine nucleotides Method HPLC Problem to be solved Diagnostics of local or somatic diseases Test material Human saliva References [133] [134] [80, 135] [82] [64] [63, 76, 86] Soils Blood Various surfaces in a hospital kitchen Water Fresh chopped meat Triclosan and toothpaste with a high fluorine content Surface of a milk tank Surfaces in food processing enterprises Microbiological quality assessment Water Freshly cut watermelon Poultry Wastewater and bottom sediments Milk Beverages (beer, carbonated beverages, and juice) Food products Bacterial count Water Poultry skin Blood Milk Porcine and bovine carcasses Urine Beer Evaluation of an environmental impact on the Soil microbial activity of soil
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Study of the effect of D,L-difluoromethylorni- Lymphatic cells of mice treated thine on mammalian cells with difluoromethylornithine Determination of enzyme activities (ATPhase and Na,K-ATPhase; glycosyltransferase) Blood Study of the role of adenine nucleotides in vas- Rabbit pulmonary artery cular physiology Determination of the freshness of fish Determination of intracellular ATP and ADP in malignant tumors as a function of cell growth Determination of microbial biomass Determination of the energy charge and equilibrium constant of adenylate kinase Bioluminescence Detection of microbiological contaminations Fish muscles

[54] [87] [55] [136] [137] [138] [139] [140] [141143] [144] [145, 146] [147] [148150] [151] [150, 152] [150] [153] [33, 154, 155] [156] [157] [157159] [160] [161] [162] [24] [163]

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METHODS FOR THE DETERMINATION OF ADENOSINE TRIPHOSPHATE Table 4. (Contd.) Method Problem to be solved Determination of the amount of active biomass Determination of the mechanism of ATP release Test material Water purification filters Aquatic ecosystem Epithelium

669

References [164, 165] [166] [167] [168]

Study of the susceptibility of bacterial strains Bacterial strains to antimicrobial agents Analysis of the susceptibility of tuberculous cells to drugs Detection of viable fungal spores on documents Analysis of the viability of the antituberculosis vaccine (BCG) Document surfaces BCG vaccine

[14] [169] [23] [170]

Determination of the suitability of blood for Blood transfusion Evaluation of the viability of cells before transplantation Study of ATP concentration changes in the storage of potatoes Determination of ATP sulfurylase activity Determination of antibiotics Milk Evaluation of sperm functions Study of ATP synthesis in mitochondria Control of hydrocarbon biodegradation Study of the role of ATP in apoptosis and necrosis Study of the effect of anticancer drugs on the growth of malignant cells Study of cell reproduction and cytotoxicity Comparison of cytostatic drugs in the treatment of lymphocytic leukemia Estimation of the number of bacterial and animal cells in a mixed population Fluorimetry Spectrophotometry Radioactivation analysis Sensors Evaluation of ADP/ATP carrier deficiencies Human muscles in human muscles Determination of phosphodiesterase activity and phosphodiesterase inhibitors Study of septic kidney injury and the monitoring of renal bioenergetics in large mammals during a septic shock Determination of the ability of ATP to regulate insulin production by an autocrine mechanism Determination of protein kinase activity
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Potato Phaseolus vulgaris extracts Sperm Mitochondria Soil

[171] [172] [173] [174] [175] [176178] [179] [180] [181, 182] [183] [184] [185] [186] [187]

Kidneys Surface of pancreatic cells

[121] [188]

[189]

[118]

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ACKNOWLEDGMENTS This study was supported by the VEGA (project no. 1/4450/07) Agency of the Ministry of Education of the Slovak Republic and the Slovak Academy of Sciences. S.V. Khlyntseva acknowledges the support of the International Visegrad Fund and the National Scholarship Programme for the Support of Mobility (SAIA). REFERENCES
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