Intracellular energy levels mainly depend on ATP, which is synthesized by mitochondria. The cell metabolome is the main pathway by which energy is supplied to cells; in addition, it provides building blocks to the cells and is correlated with cell signaling ^[1-4]. Most biochemical reactions are linked with ATP-ADP conversion. Many persistent organic pollutants, including quinones, have strong effects on intracellular metabolites ^[5-10]. Every 1-2 min, nearly 5 pg of ATP is used in each cell, thus, approximately 65 kg of ATP is hydrolyzed in the body each day. ATP levels remain constant, thus, the same amount of ATP is produced as is consumed ^[11]. This study provides an alternative method for examining the action of metabolic pathways by quantifying the formation/consumption of ATP/ADP during biochemical reactions and is thus of great significance.

The structure and physicochemical properties of ATP, ADP, AMP are very similar, rendering the accurate quantification of these substances difficult. Thus, many different methods, including those based on aptamers ^{[12, 13]}, sensors ^[14-17], HPLC ^[18-25] and nuclear magnetic resonance spectroscopy ^[26] have been studied for the quantification of ATP and its metabolites in biological fluids, herbal materials, and foods. However, these methods require long analysis times, and the system is unstable. In the most commonly used HPLC-based methods ^[18-25], the retention time differs greatly between peaks. In addition, the retention time is less than 5 min, but the retention times of a variety of small molecules in the cell are also near this value, which renders interpretation difficult.

We conducted an initial literature search, and based on previous research, this study succeeded in quantifying intracellular ATP, ADP, and AMP contents. In addition, the effect of quinones on ATP, ADP, AMP levels in MRC-5 cells was studied, and the results showed correlations between levels of the three metabolites and TBBQ concentration.

1 Experimental

1.1 Materials

ATP, ADP and AMP standards and disodium hydrogen phosphate (Na₂HPO₄\512H₂O) of analytical grade were purchased from Sigma, USA. Methanol was purchased from Fisher Scientific (Thermo, USA). Acetic acid (HAc) was purchased from Sinopharm Chemical Reagent Beijing Co., Ltd., China. The methanol and water used in this study were passed through a 0.22 μ m filter before use.

1.2 Cell culture and treatment

A human fetal lung fibroblast cell line (MRC-5) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing high glucose, which contained 10% (v/v) fetal bovine serum, 100 g/L streptomycin, and 100 U/mL penicillin under an atmosphere of 5% (v/v) CO₂ at 37 ℃. After culturing for 24 h, the MRC-5 cells were treated with 10, 20, and 50 mmol/L 2-tert-butyl-1, 4-benzoquinone (TBBQ). At the concentration of 50 mmol/L, the TBBQ can cause MRC-5 cell death. After treatment for 24 h with TBBQ, the cells were harvested for further analysis.

The harvested cells were counted using a Handheld Automated Cell Counter (Millipore, USA) and treated with 200 μ L of 80% (v/v) ice-cold methanol. After centrifuging the extracted mixture at 12 000 r/min for 5 min at 4 ℃, the supernatant was decanted, subjected to ultrafiltration, and centrifuged at 12 000 r/min for 20 min at 4 ℃. Finally, 200 μ L of the solvent A (50 mmol/L disodium hydrogen phosphate and 15 mmol/L trimethylamine (TEA), pH 7.88 adjusted using acetic acid (HAc)) was added.

1.3 HPLC analysis

All prepared samples were analyzed using a Shimadzu HPLC system. An Agela Venusil MP C18 column (250 mm×4.6 mm, 5 μ m) was used. The mobile phases comprised solvent A and solvent B (methanol). Ion-pair reversed-phase HPLC was used to separate the compounds of interest in the cultured cells. Isocratic elution (4% (v/v) methanol) was used. The flow rate used in this study was 0.8 mL/min, and the injection volume used for all samples and standards was 20 μ L. Ambient temperature and dual-wavelength spectrophoto-metry were used in this study. Analyte peaks were recorded at 254 nm and 266 nm.

2 Results and discussion

2.1 Qualitative and quantitative analysis

The retention times of ATP and its metabolites (ATP, ADP, and AMP standards) were determined using our method (Fig. 1a). The retention times were as follows: ATP, 15.24 min; ADP, 16.54 min; AMP, 13.70 min. The resolutions obtained (R_ATP/AMP=1.8, R_ATP/ADP=1.4) show that the method developed in this study can be used to quantify the three metabolites and that the results are reliable. Then, we detected the intracellular levels of ATP, ADP, AMP successfully (Fig. 1b) using our developed protocol.

Fig. 1

Fig. 1 Qualitative analysis of ATP, ADP, AMP and their quantification in MRC-5 cells a. the sample extracted from MRC-5 cells using the described protocol; b. 10 μ mol/L mixed standard sample of AMP, ADP and ATP; c-e. 10 μ mol/L standard samples of ATP, ADP and AMP.

2.2 Optimum chromatographic conditions

The effect of pH on the system was studied by adjusting the pH to 6.08, 7.03 and 7.88. Methanol was used as the organic eluent. Fig. 2a illustrates the separation behavior at pH 6.08, 7.03 and 7.88. The eluent pH significantly affected the separation of ATP and the two metabolites. The results clearly show that ATP, ADP and AMP are resolved most clearly at pH 7.88.

Fig. 2

Fig. 2 Effects of different chromatographic conditions a. pH; b. flow rate; c. methanol volume percentage; d. equilibrium time. Peaks: 1. AMP; 2. ATP; 3. ADP.

The influence of flow rates (Fig. 2b) and the concentrations of methanol (Fig. 2c), TEA and Na₂HPO₄ were investigated. Various TEA and Na₂HPO₄ concentrations were studied, and other conditions were kept equal. The results showed that the resolution was highest at 15 mmol/L TEA, 50 mmol/L Na₂HPO₄ and 4% (v/v) methanol, with a flow rate of 0.8 mL/min. In addition, the results in Fig. 2d indicated that the equilibrium time could be shortened to 30 min (comparing with previous studies ^[19]).

Under the optimal experimental conditions, we obtained a working curve using the developed method. The following results were obtained: linear regression equation for ATP was Y=2 147.65+18 275.28C with correlation coefficient R²=0.999 9; linear regression equation for ADP was Y=221.72+18 739.93C with R²=0.999 9; linear regression equation for AMP was Y=-8 580.03+20 792.43C with R²=0.999 6 (Y: peak area; C: concentration, μ mol/L.) The linear range for all compounds was approximately from 0.1 to 100 μ mol/L.

2.3 Application to actual samples and effects of TBBQ on metabolite levels

The developed HPLC method was then applied to the quantification of ATP and its two catabolites in cultured MRC-5 cells. MRC-5 cells had been seeded in 10 cm plates containing DMEM/high glucose medium one day before the experiment. The cells were treated with TBBQ for 24 h with different concentrations. Subsequently, ATP, ADP, AMP levels and their ratios were determined as described previously. The results (Fig. 3) indicated that TBBQ could affect the concentrations of ATP, ADP. ATP level was decreased when the concentration of TBBQ was 10 mmol/L but increased when the concentration was 20 mmol/L. ADP level decreased as the concentration of the TBBQ increasing; AMP levels kept constant with increasing level of TBBQ.

Fig. 3

Fig. 3 Effects of TBBQ on the levels of ATP, ADP and AMP in MRC-5 cells The concentrations of each pollutant were (a) 10 mmol/L and (b) 20 mmol/L.

3 Conclusions

Here, we presented an HPLC-based method for the separation and quantification of ATP and its two metabolites in cells using TEA and Na₂HPO₄ as ion-pair reagents. The developed method provided high sensitivity and selectivity over a wider linear concentration range and proved more stable than the previously reported ion-pair reversed-phase HPLC method. Next, using the validated method, intracellular ATP and its two metabolites were successfully detected in MRC-5 cells. We concluded that the developed approach was generally applicable to the determination of intracellular metabolites in actual samples. We also explored the effects of TBBQ on the three analytes. The results showed that TBBQ additives could rescue intracellular ATP levels when it was in the higher concentration, indicating that TBBQ could cause metabolic disorders in cells and even in the body.

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