Adenosine 5′-diphosphate

Intracellular adenosine 5 -triphosphate, adenosine 5 -diphosphate, and adenosine 5 -monophosphate detection by short-end injection capillary electrophoresis using methylcellulose as the effective electroosmostic flow suppressor

Keywords:
Adenosine 5 -diphosphate / Adenosine 5 -monophosphate / Adenosine 5 -triphos- phate / Capillary electrophoresis /Methylcellulose

1 Introduction

Intracellular adenosine 5 -triphosphate (ATP) is the most important source of cellular energy which is liberated dur- ing its transformation into adenosine 5 -diphosphate (ADP). Together with ATP, ADP, and adenosine 5 -monophosphate (AMP) form the adenylate system [1]. ATP is produced as an energy source during the processes of photosynthesis and cellular respiration and consumed by many enzymes and a multitude of cellular processes including biosynthetic reac- tions, motility, and cell division [2]. In signal transduction pathways, ATP is used as a substrate by kinases which

Correspondence: Dr. Angelo Zinellu, Department of Biomedical Sciences, Chair of Clinical Biochemistry, University of Sassari, Viale San Pietro 43/B, I-07100, Sassari, Italy

Abbreviations: ADP, adenosine 5 -diphosphate; AMP, adenosine 5 -monophosphate ; ATP, adenosine 5 -triphosphate; G6PD, glu- cose-6-phosphate dehydrogenase; HK, hexokinase; MC, methyl- cellulose

phosphorylate proteins and lipids, as well as by adenylate cyclase, which uses ATP to produce the second messenger molecule cyclic AMP [3,4]. Moreover, extracellular ATP acti- vates cell-surface receptors of P2 type, divided in two sub- groups called transmitter-gated ion channel P2X receptors, permeable in the open state to sodium, potassium and cal- cium, and metabotropropic P2Y receptors coupled to G proteins [5]. Therefore, the adenylate system plays an essential role in all living organisms and the proper balance between [ATP], [ADP], and [AMP] is fundamental to keep the cellular energy state within a narrow range to maintain the homeostasis. However, various kinds of metabolic stress can cause the alteration of the energy charge which may fall below the physiological range [6–11]. Therefore, to evaluate the levels of adenine nucleotides in biological matrices sev- eral methods have been developed. The separation and analysis of intracellular nucleotides were first performed by HPLC [12]. However, while resolution is generally adequate, run times with HPLC are fairly long, e.g., 30–45 min. As an alternative for this type of analysis, CE in the CZE [13, 14] or MECC [15] modes has been proposed. Nucleotides are easily resolved in CE thanks to their negative charge in a wide pH range (from 2 to 12). However, also in this case elevated run times are necessary to separate the three phosphate forms due to the high differences in their charge density. In order to reduce the analysis time in CE, short total capillary lengths [16–19] and/or high voltages [17] are commonly recommended. However, in these approaches high currents may be generated with a loss of efficiency and reproducibility [19]. The analysis time can also be reduced using a short-end injection procedure where the separation length is decreased by injecting the sample at the capillary end closest to the detector window (outlet). Thus, the migration distance of analytes is significantly reduced (10.2 cm in this study) allowing shorter migration times. The application of the short-end injection for separation was first introduced by Mazzeo and Krull [20] for the protein analysis by CIEF. Melanson and Lucy [21] have recently w/v TCA. After centrifugation (30006gfor 5 min), 50 mL of supernatant was mixed with 150 mL of distilled water and subsequently injected in CE.

2.3 Extraction of adenine nucleotides from spermatozoa and enzymatic measurement of intracellular ATP

Semen was obtained by an artificial vagina from adult bucks (weight: 41–44 kg, aged between 5 and 8 years) maintained in an outdoor environment and fed a live-weight main- tenance ration.
Fifty microliters of collected spermatozoa (approximately 1.1610 cells/mL) were washed twice with 0.1 mL of cold physiological solution. For the extraction of nucleotides, 0.1 mL of ice-cold 0.6 mol/L perchloric acid were added to each Eppendorf containing spermatozoa and kept for 15 min; after the suspension was centrifuged in an Eppendorf micro- fuge (3 min at 10 000 rpm) and the supernatant was neu-free-solution CZE that allows the analysis separation for nitrite and nitrate in about 12 s. We have recently proved the applicability of the short-end injection mode in the clinical field setting up a new method for a rapid analysis of serum creatinine (migration times 70 s) [22]. By this new work we have developed a new rapid CE method for the quantification of ATP, ADP, and AMP levels in cells. The method applicability was proved by measuring analytes concentration in red blood cells and in bucks sperm. To evaluate the accuracy of CE analysis, the data obtained by our proposed method and by a reference enzymatic assay for ATP quantification were compared by statistical Pas- sing–Bablok regression and the Bland–Altman test.

2 Matherials and methods

2.1 Chemicals

ATP, ADP, AMP, glacial acetic acid, sodium acetate, hydro- chloric acid, K2CO3, perchloric acid, glucose-6-phosphate dehydrogenase (G6PD), hexokinase (HK), glucose, nicotina- mide adenine dinucleotide phosphate, TRAP buffer, and methylcellulose (MC) were from Sigma –Aldrich. All chemi- cals used were of analytical reagent grade. Membrane filters (0.45 mm) used to filter all buffer solutions before CE analy- sis and Microcon-10 microconcentrators (cutoff Mr 10 000) were obtained from Millipore (Bedford, MA, USA).

2.2 Extraction of adenine nucleotides from red blood cells

Red blood cells were separated from plasma by centrifuga- tion (50006gfor 5 min) followed by three washings with 0.9% w/v NaCl and were immediately processed. Thawed packed cells (100 mL) were lysed by adding 200 mL of 7.5%

sive centrifugation in a Microfuge (3 min at 10 000 rpm), the supernatant was analyzed spectrophotometrically with enzy- matic assay and by CE. For the enzymatic assay ATP levels were measured spectrophotometrically at 340 nm using NADH-linked enzyme-coupled assays [24] modified for being adapted to our system. The enzymatic assay was carried out at 377C with a Beckman DU-7 spectrophotometer, and per- formed using the coupling enzymes, G6PD and HK. Per- chloric extract (25 mL) were added to 400 mL of TRAP buffer (0.1 mol/L, pH 7.6). To the mixture also 2 mL of HK (2 mg/ mL), 2 mL of G6PD (1 mg/mL), 8 mL of glucose (18 mg/mL), and 8 mL of nicotinamide adenine dinucleotide phosphate (NADP ) (20 mg/mL) were added. ATP was determined from the production of NADPH.

2.4 CE analysis

An MDQ CE system equipped with a DAD was used (Beck- man Instruments, Fullerton, CA, USA). The system was fit- ted with a 30 kV power supply with a current limit of 300 mA. Analysis was performed in an uncoated fused-silica capillary (75 mm id and 30 cm total length), injecting 20 nL of sample (5 s at 0.5 psi) at the outlet end. Separation was carried out in a 60 mmol/L sodium acetate buffer pH 3.8, MC 0.01%, 157C, and 17 kV (90 mA) at normal polarity (with the anode at the outlet). After each run, the capillary was rinsed with 1 min of 0.1 mmol/L HCl and equilibrated with run buffer for 1 min. Separation was monitored at 254 nm wavelength.

3 Results and discussion

ATP, ADP, and AMP may be easily resolved in CE due to their negative charge in a wide pH range. However, owing to the considerable difference in charge density between these analytes their migration times could significantly differ yielding long analytical times, as shown in Fig. 1A. In this case, since the analysis was performed in a 60 mmol/L sodium acetate run buffer (pH 3.8) at reverse polarity, the most charged analytes migrated towards the anode faster than the less charged ones. Thus, the migration order was ATP, ADP, and AMP with this last analyte taking about 30 min to reach the detection windows sited at 20 cm from the inlet. To reduce the migration time of AMP we attempted to minimize the EOF, which in the adopted electrophoretic conditions was opposite to the migration direction of ana- lytes and though at pH 3.8 its intensity was low, it delayed the AMP migration. As previously described [25], we mini- mized the EOF by adding MC (0.01% final concentration) to the run buffer, thus decreasing the migration time of AMP to about 10 min (Fig. 1B). To further cut the run time, we reduced the migration distance as much as possible injecting the analytes at the end of the capillary, near to the detection window, under the same electrophoretic conditions (the polarity of the electrodes was obviously switched over). In this case, the migration distance was reduced to 10 cm and the run time was halved (Fig. 1C). In order to evaluate the best wavelength for the analytes detection we performed an absorbance spectra between 190 and 300 nm in the selected run buffer. As shown in the inset of Fig. 1C, ATP had the highest molar coefficient extinction at 204 and 254 nm. Similar results were observed for ADP and AMP (data not shown) so that the whole analysis was monitored at 254 nm. In Table 1 we compared analysis time, peak height and effi- ciency of peaks between the normal injection mode with and without MC and the short-end injection mode in presence of MC (Table 1). In particular it is evident that the run time decreased of about six times when reverse injection with MC was used and the peak height raised, thus also increasing the method sensitivity ( 136% for ATP, 151% for ADP, and 1346% for AMP). We applied the new CE method for the evaluation of adenine nucleotides in red blood cells and in particular, as first application, we investigated the effect of red blood cell treatment on ATP, ADP and AMP recovery. Figure 2 shows the electropherograms of adenine nucleo- tides obtained from erythrocytes lysed with 7.5% TCA (A) and with water (B). As evident by the graph, the water lysis determined a loss of ATP with increased levels of ADP and AMP due to the ATPase activity that is instead suppressed by acidic treatment. However, if the samples were maintained

Figure 1. Electropherograms of standard adenine nucleotides. Electrophoretic conditions: (A) 60 mmol/L sodium acetate pH 3.8, 20 cm effective capillary length (30 cm total length), inlet injec- tion, and reverse polarity. (B) 60 mmol/L sodium acetate pH 3.8, 0.01% MC, 20 cm effective capillary length (30 cm total length), inlet injection, and reverse polarity. (C) 60 mmol/L sodium ace- tate, 10 cm effective capillary length (30 cm total length), outlet injection, and normal polarity. For all: 157C cartridge tempera- ture, 17 kV applied voltage, 254 nm detection wavelength. The inset of panel (C) shows the absorbance spectra for ATP obtained by DAD of CE between 190 and 300 nm in the selected run buffer. Similar results were observed for ADP and AMP (data not shown).

Table 1. Comparison between some electrophoretic parameters obtained by normal and reverse injection with
and without MC

Migration time (min) Peak height (au) Efficiency (N/m)
ATP ADP AMP ATP ADP AMP ATP ADP AMP

Normal 3.03 4.60 26.76 52 830 50815 18 095 9 204 9 865 3 367
Normal 1MC 2.62 3.67 10.14 46 519 49969 51 773 13 687 17 342 25 134

Figure 2. Electropherogram of adenine nucleotides from red blood cells lysed with 7.5%TCA (A) and in water (B). Electropho- retic conditions: as in Fig. 1C.

for 24 h at 237C in a TCA solution a degradation of ATP and an increase of ADP and AMP levels (perhaps due to the ATP breakdown) could be evidenced (data not shown). Therefore, even if red blood cell lysis by TCA is advisable for a better quantification, the analysis must anyhow be performed immediately to avoid pitfalls. As reported in Fig. 3, our method is also able to detect ATP, ADP, and AMP in more complex samples like spermatozoa.
The linearity of the detector response to different analyte concentrations was determined between 20 and 1600 mmol/ L for ATP and between 20 and 200 mmol/L for ADP and AMP. The calibration curves Y = 8.9X 2 14.3 (ATP) , Y = 10X 14.9 (ADP), and Y = 6.5X 16.6 (AMP) show linear responses over the concentration tested with regression coefficients R = 0.999 for all the analytes. Within run preci- sion (intra-assay) of the method was evaluated by injecting the same plasma pool ten times consecutively, while be- tween-run precision (interassay) was determined by inject- ing the same sample in ten consecutive days. Precision tests indicated a good repeatability of the method (intra-assay CV,4%,interassay CV,8%.For the assessment of the ana- lytical recovery, biological samples were spiked with standard solutions and the means of recovery, evaluated by five differ- ent experiments were 98.3% for ATP, 99.0% for ADP, and 98.6%for AMP. To determine the lowest LOD, serial dilu- tions (in water) of a mix standard were injected and the con- centrations giving the smallest observable peak were identi-

Figure 3. Electropherogram of adenine nucleotides from sper- matozoa sample. Electrophoretic conditions as in Fig. 1C.

fied: the LODs for an S/N of 3 were 6 mmol/L for ATP and ADP and 5 mmol/L for AMP. The accuracy of the new CE method was assessed by comparison to a reference spectro- photometric assay. Forty spermatozoa samples were analyzed and adenine nucleotides concentrations were determined. The results were evaluated by specific statistical methods for the measurement comparison. In particular, the Bland–Alt- man test, obtained by plotting the differences between the analyte concentrations measured by the two assays against the average of the two values, demonstrated the absence of a systematic bias (Fig. 4A). Passing and Bablok regression analysis showed a linear relationship between the two meth- ods and a close agreement of both slop and intercept with the target values of 1 and 0 within the 695% confidence limits (Fig. 4B). Moreover, the Cusum test for linearity showed no significant deviation from linearity ( p .0.05)for all the tested analytes. The statistical comparisons indicated that the data obtained by the two methods are equivalent.

4 Concluding remarks

ATP is a ubiquitous, energy-rich compound in all cells of living organisms. It is found both within organelles, such as mitochondria and chloroplasts, and in the cytoplasm of higher organisms. The energy derived from ATP is used to drive a multitude of vital biochemical reactions that are fun- damental to the survival of cells and the whole organism. The energy content of cells depends upon the balance be- tween the production and the consumption of energy so that the levels of ATP, ADP, and AMP are tightly regulated. However, some metabolic stress can cause the alteration of intracellular energy charge which may fall below the physio- logical range. Therefore, methods able to measure adenine nucleotides are needed to monitor the energy state of cells. ATP, ADP, and AMP have commonly been measured by using bioluminescence reagents containing firefly luciferase [26, 27], though HPLC [12], or CE [13–15] methods have been also described. In all these cases tedious preanalytical proce- dures or long analytical times are required to perform the analysis. By this work we propose a new CE method with rapid sample preparation and fast analytical times guaran- teed by the short-end injection mode and by the use of MC as EOF suppressor. We have shown the advantages of our pro- posed procedure which allows an improvement in analyses times (5 min instead 30 min needed with the normal injec- tion) and in sensitivity (peak height increase from 36 to 350% compared to those obtained with the normal config- uration). The application of our method has been proved both on red blood cells and spermatozoa. This last applica- tion may have a particular importance since the ATP sper- matozoa levels are related to value mitochondrial integrity after cryopreservation, thus potential applications for assist- ed reproductive technologies are also possible.

This study was supported by the “Fondazione Banco di Sar- degna, Sassari, Italy”and by the “Ministero dell’Università e della

CE and CEC 3073

Ricerca,”Italy. The manuscript language revision by Mrs. Maria Antonietta Meloni is greatly appreciated.

The authors have declared no conflict of interest.

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