ZHU Huiyu, WU Danni, WANG Hailin(,-,,, 100085,)

Special issue for commemorating Professor ZOU Hanfa (Ⅱ)·Article

Quantification of intracellular adenosine 5′-triphosphateand its metabolites by high performance liquidchromatography analysis

ZHU Huiyu, WU Danni, WANG Hailin*
(StateKeyLaboratoryofEnvironmentalChemistryandEcotoxicology,ResearchCenterforEco-EnvironmentalSciences,ChineseAcademyofSciences,Beijing, 100085,China)

This study was aimed to provide insight regarding the intracellular metabolites of adenosine 5′-triphosphate (ATP) and whether 2-tert-butyl-1,4-benzoquinone (TBBQ) affects cell metabolites. A rapid high performance liquid chromatography (HPLC) protocol was developed for the separation and quantitation of ATP and its metabolites (adenosine diphosphate (ADP) and adenosine monophosphate (AMP)) in cells. Chromatographic separation was performed using a Shimadzu HPLC system equipped with an Agela Venusil MP C18 column; isocratic elution was adopted. The mobile phase comprised solvent A (50 mmol/L disodium hydrogen phosphate and 15 mmol/L trimethylamine (TEA); pH adjusted to 7.88 using acetic acid (HAc)) and solvent B (methanol). The correlation coefficients of the three analytes were very high (R2≥0.999 6), and the contents of the three metabolites in the MRC-5 cells were within the linear ranges (0.1-100 μmol/L). The limits of detection for the detected three compounds were low. Samples were extracted from cells (after exposure and non-exposure to quinones) using 80% (v/v) methanol aqueous solution. The method developed in this study was successfully applied to detect ATP, ADP and AMP in MRC-5 cells, and the results demonstrated that ATP, ADP, AMP levels in cells were affected by TBBQ, but the relations between the concentration of TBBQ and the level of ATP, ADP and AMP were complex.

high performance liquid chromatography (HPLC); adenosine 5′-triphosphate (ATP); quinones; metabolites; intracellular

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 (Na2HPO4512H2O) 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 μm 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) CO2at 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 μL 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 μL 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 μm) 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 μL. Ambient temperature and dual-wavelength spectrophoto-metry were used in this study. Analyte peaks were recorded at 254 nm and 266 nm.

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.

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 (RATP/AMP=1.8,RATP/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 Qualitative analysis of ATP, ADP, AMP andtheir quantification in MRC-5 cells a. the sample extracted from MRC-5 cells using the described protocol; b. 10 μmol/L mixed standard sample of AMP, ADP and ATP; c-e. 10 μmol/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.

The influence of flow rates (Fig. 2b) and the concentrations of methanol (Fig. 2c), TEA and Na2HPO4were investigated. Various TEA and Na2HPO4concentrations were studied, and other conditions were kept equal. The results showed that the resolution was highest at 15 mmol/L TEA, 50 mmol/L Na2HPO4and 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 wasY=2 147.65+18 275.28Cwith correlation coefficientR2=0.999 9; linear regression equation for ADP wasY=221.72+18 739.93CwithR2=0.999 9; linear regression equation for AMP wasY=-8 580.03+20 792.43CwithR2=0.999 6 (Y: peak area;C: concentration, μmol/L.) The linear range for all compounds was approximately from 0.1 to 100 μmol/L.

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.

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.

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 Na2HPO4as 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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朱會宇, 吳丹妮, 汪海林*
(中國科學(xué)院生態(tài)環(huán)境研究中心, 環(huán)境化學(xué)與生態(tài)毒理學(xué)國家重點(diǎn)實(shí)驗(yàn)室, 北京 100085)

研究了三磷酸腺苷(ATP)及其代謝物在細(xì)胞內(nèi)的含量以及2-叔丁基-1,4-苯醌(TBBQ)對ATP及其代謝產(chǎn)物在細(xì)胞內(nèi)含量的影響。建立了一種高效液相色譜法(HPLC)用于快速分離、檢測細(xì)胞內(nèi)ATP及其代謝產(chǎn)物(二磷酸腺苷(ADP)和一磷酸腺苷(AMP))的含量。使用島津高效液相系統(tǒng)及艾杰爾Venusil MP C18柱,采用等度洗脫的方式。流動相A相為50 mmol/L磷酸氫二鈉和15 mmol/L三甲胺(TEA),用醋酸(HAc)調(diào)節(jié)pH至7.88;流動相B相為甲醇。采用建立的高效液相色譜法得到了3種代謝物的工作曲線,相關(guān)系數(shù)高(R2≥0.999 6), MRC-5細(xì)胞中3種代謝物的含量均在線性范圍(0.1～100 μmol/L)內(nèi)。該方法檢出限低。采用預(yù)冷的80%(體積分?jǐn)?shù))甲醇水溶液提取細(xì)胞內(nèi)的代謝物。該研究建立的方法成功地應(yīng)用于檢測MRC-5細(xì)胞中的ATP、ADP和AMP的含量,結(jié)果表明,TBBQ會對ATP、ADP、AMP在細(xì)胞內(nèi)的含量產(chǎn)生影響,但TBBQ濃度和ATP、ADP以及AMP在MRC-5細(xì)胞內(nèi)濃度的關(guān)系比較復(fù)雜。

高效液相色譜;三磷酸腺苷;醌類;代謝產(chǎn)物;細(xì)胞內(nèi)

10.3724/SP.J.1123.2016.08031

Foundation item: National Natural Science Foundation of China (No. 21327006).

O658

: AArticle IC:1000-8713(2017)01-0054-05

高效液相色譜法測定細(xì)胞內(nèi)三磷酸腺苷及其代謝物的含量

*Received date: 2016-08-26

*Corresponding author.Tel: +86-10-62849010, E-mail: hlwang@rcees.ac.cn.