Razvijena je jednostavna i brza metoda određivanja masenog udjela adenozin trifosfata (ATP), adenozin difosfata (ADP) i adenozin monofosfata (AMP) visokodjelotvornom tekućinskom kromatografijom (HPLC) u perikarpu voća liči. Pri određivanju adenozin fosfata upotrijebljen je acetonitril koji skraćuje vrijeme postupka. Također je postignuta dobra ponovljivost (koeficijent varijacije iznosi 1,28-1,80 %) i obnovljivost (94,7-97,1 %). Koeficijenti korelacije s površinama pika u rasponu od 0 do 80 ng iznosili su 0,9946 za ATP; 0,9994 za ADP i 0,9974 za AMP. Ova je metoda primijenjena za određivanje masenog udjela adenozin fosfata u perikarpu voća liči pri berbi, pa je utvrđeno da je maseni udio ATP-a iznosio 27,4 μg/g, ADP-a 35,4 μg/g i AMP-a 7,9 μg/g, na bazi svježe tvari voća.A simple and rapid method using high performance liquid chromatography (HPLC) was developed to determine levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) in litchi fruit pericarp tissue. This HPLC method used acetonitrile gradient elution and shortened the time required for determinations of adenosine phosphates. This analysis exhibited good repeatability (coefficients of variation 1.28–1.80 %) and recovery rate (94.7–97.1 %). The correlation coefficients of ATP, ADP and AMP with their peak areas at a range of 0–80 ng were 0.9946, 0.9994 and 0.9974, respectively. This method was applied to determine levels of adenosine phosphates in pericarp tissue of litchi fruit at harvest. There were 27.4 μg/g of ATP, 35.4 μg/g of ADP and 7.9 μg/g of AMP on a fresh mass basis

Hai Liu

Weibo Jiang

Yueming Jiang

Yunbo Luo

Food Technology and Biotechnology

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ISSN 1330-9862 scientific note(FTB-1485)A Simple and Rapid Determination of ATP, ADP and AMPConcentrations in Pericarp Tissue of Litchi Fruit by HighPerformance Liquid ChromatographyHai Liu1,3, Yueming Jiang1*, Yunbo Luo2 and Weibo Jiang21South China Botanic Garden, The Chinese Academy of Science,510650 Guangzhou Leyiju, PR China2College of Food Science and Nutritional Engineering, China Agricultural University,Qinghuadonglu, 100083 Beijing, PR China3Graduate School of the Chinese Academy of Science, 100039 Beijing, PR ChinaReceived: February 25, 2005Accepted: May 25, 2005SummaryA simple and rapid method using high performance liquid chromatography (HPLC)was developed to determine levels of adenosine triphosphate (ATP), adenosine diphos-phate (ADP) and adenosine monophosphate (AMP) in litchi fruit pericarp tissue. This HPLCmethod used acetonitrile gradient elution and shortened the time required for determina-tions of adenosine phosphates. This analysis exhibited good repeatability (coefficients ofvariation 1.28–1.80 %) and recovery rate (94.7–97.1 %). The correlation coefficients of ATP,ADP and AMP with their peak areas at a range of 0–80 ng were 0.9946, 0.9994 and 0.9974,respectively. This method was applied to determine levels of adenosine phosphates in pe-ricarp tissue of litchi fruit at harvest. There were 27.4 mg/g of ATP, 35.4 mg/g of ADP and7.9 mg/g of AMP on a fresh mass basis.Key words: HPLC, ATP, ADP, AMP, determination, litchi fruit, pericarp tissueIntroductionEnergy metabolism is an important metabolic path-way which maintains biochemical and physiological ac-tivities in plant tissues (1). Energy charge can be used toshow energy status in plant tissues (1,2), while synthe-sis, degradation and restoration of membrane lipids de-pend on energy supply (3–5). The energy charge of cellscan be calculated as ([ATP] + 0.5 [ADP])/([ATP] + [ADP]+ [AMP]), as described by Pradet and Raymond (1). Someof key enzymes concerning glycolysis, the Krebs’ cycle,the electron transport system and oxidative phosphory-lation are also regulated by the energy level of cells (6,7).Lipids are the essential components of plant cell mem-branes, and involvement of adenylate nucleotides infatty acid biosynthesis in membrane lipid is well estab-lished (5,8). Thus, energy status of tissues of fruits andvegetables may play an important role in maintainingthe integrity of cell membranes, which is related to post-harvest life (7,9).The luciferase system was mainly used to determineATP concentration in organisms, while the analysis ofATP and ADP was performed by enzyme-linked immu-nosorbent assay (10–12). A high performance liquid chro-matography (HPLC) analysis of ATP and its degrada-tion products was also developed on reverse-phase col-umns using phosphate buffer as a mobile phase (13,14).Organic solvents, such as methanol (15,16) or acetonitrile(14), can be used to reduce the running time. In practice,these methods need a long running time and it is neces-sary to develop a simple and rapid method to analyze alarge number of fruit samples (14,17,18).531H. LIU et al.: Determination of ATP, ADP and AMP in Litchi Fruit, Food Technol. Biotechnol. 44 (4) 531–534 (2006)*Corresponding author; Phone: ++86 20 37 252 905; Fax: ++86 20 37 252 831; E-mail: ymjiang@scib.ac.cnThere are no reports on the determinations of ATP,ADP and AMP contents by HPLC in plant tissue. Theobjective of this study was to develop a simple and ra-pid HPLC method for measurements of ATP, ADP andAMP content in pericarp tissue of litchi fruit.Materials and MethodsReagentsThe high purity ATP, ADP and AMP standards, per-chloric acid, potassium dihydrogen phosphate, potassi-um hydrogen phosphate and acetonitrile (HPLC grade)were purchased from Sigma Chemical Co. All reagentsused were dissolved in deionized water, and then filte-red with the Sybron/Barnstead Millipore system and a0.45-mm filter paper.Plant materialsFruit of litchi (Litchi chinensis Sonn. cv. Huaizhi) atthe commercially mature stage was harvested from anorchard in Guangzhou. Litchi pericarp was collected forextraction and determinations of ATP, ADP and AMP.Preparations of standard stock solutionsATP, ADP and AMP standards (1 mg) were eachdissolved in 10 mL of deionized water to obtain ATP,ADP and AMP standard stock solutions at 100 mg/mL.Aliquots of the stock standard solutions were made bydiluting them in deionized water at 0, 0.2, 0.5, 1, 2 and 4mg/mL. A volume of 20 mL of each sample was takenfor HPLC analysis.Mobile phasesMobile phase A consisted of 0.06 mol/L dipotassi-um hydrogen phosphate and 0.04 mol/L potassium di-hydrogen phosphate dissolved in deionized water andadjusted to pH=7.0 with 0.1 mol/L potassium hydrox-ide, while mobile phase B consisted of 100 % acetoni-trile. Air bubbles in the two solutions were driven awayusing an ultrasonic instrument.Extraction of ATP, ADP and AMP from litchifruit pericarp tissueLitchi fruit pericarp tissue (2 g) was rapidly frozen inliquid nitrogen and homogenized into powder. Adeno-sine phosphates were extracted from the powder with10 mL of 0.6 mol/L perchloric acid in the ice bath for 1min by the method of Yang et al. (12). The extractionmixture was centrifuged for 10 min at 6 000´g (BeckmanJ20-2) and 4 °C, and 6 mL of the supernatant was takenand quickly neutralized to pH=6.5–6.8 with 1 mol/L KOHsolution. The neutralized supernatant was then allowedto stand for 30 min in an ice bath to precipitate most ofthe potassium perchlorate, which was removed by paperfiltration. The filtrate solution was filtered again througha 0.45-mm filter. The final filtrate solution was made upto 8 mL and then stored at –30 °C prior to the analysis.HPLC analysisThe HPLC (Gold 125 Solvent System, Beckman In-struments Inc., USA) conditions were as follows: anUltrasphere ODS EC 250´4.60 mm column (Beckman In-struments Inc., USA) was equipped with a Beckman 125pump system. Peaks were detected and analyzed at 254nm by a Gold 168 diode array detector. HPLC separa-tion was achieved using continuous gradient elution.The elution program was as follows: 0 min 100 % A, 0 %B; 2 min 95 % A, 5 % B; 4 min 80 % A, 20 % B; 5.3 min75 % A, 25 % B and 6 min 100 % A, 0 % B. Finally, theprogram took a further 1 min to return to the initial con-ditions and stabilize. Flow rate of the mobile phase was1.2 mL/min, while the injection volume was 20 mL. Thetotal retention time was about 5 min and the gradientwas run for 6 min to ensure full separation. ATP, ADPand AMP in the samples were identified by comparisonwith retention time of standards, while the concentra-tions of ATP, ADP and AMP were determined using theexternal standard method. Data were expressed asmeans of six replicate determinations.Recovery trialThree standards (ATP, ADP and AMP) were addedinto litchi fruit pericarp tissue. The extraction was car-ried out according to the above-mentioned method. Theconcentration of each standard added for the trial was 2mg/mL. The whole experiment was repeated six times.Statistical analysisQuantitative data from the HPLC analysis werecompared using either the coefficients of variation (CV)or analysis of variance (ANOVA). Least significant dif-ference was used to compare the means.Results and DiscussionHPLC analysis and calibration curves of ATP,ADP and AMPATP and its breakdown products exhibited a greatabsorbance at 254 nm (13). As shown in Fig. 1, ATP,ADP and AMP were separated well and detected at 254nm in this study. Acetonitrile instead of methanol usedas mobile phase gave a rapid and better separation ofATP, ADP and AMP (data not shown) because it hashigher polarity than methanol. Ryder (13) obtained a532 H. LIU et al.: Determination of ATP, ADP and AMP in Litchi Fruit, Food Technol. Biotechnol. 44 (4) 531–534 (2006)0 1 2 3 4 5 6 70.0000.0010.0020.0030.0040.005ATPADPAMPA254nmRetention time/minFig. 1. HPLC chromatogram of a standard mixture of ATP, ADPand AMPsufficient resolution of adenosine phosphates within 16min using an isocratic system. Veciana-Nogues et al. (16)reported a good separation of adenosine phosphates us-ing 30 % methanol gradient conditions. In this study, theseparation and determination of ATP, ADP and AMPneeded only 6 min and, thus, it is very convenient to an-alyze a large number of samples.There was a good linear relationship among ATP,ADP and AMP concentrations at a range of 0–80 ngagainst their peak areas, with correlation coefficients being0.9946, 0.9994 and 0.9974, respectively, at the 5 % level.Using 2 mg/mL standards, the CV of the levels of ATP,ADP and AMP were 1.80, 1.28 and 1.30 %, respectively,all below 5.00 % (Table 1), which indicated excellent re-peatability for the analysis of the three compounds.Recovery trialHigh precision was important to determine the con-centrations of ATP, ADP and AMP (14,19). The resultsindicated that the mean recovery rates of ATP, ADP andAMP were 94.9, 94.7 and 97.1 %, respectively. These werevery high recovery rates bearing in mind the complexityof the analyses. The CV of ATP, ADP and AMP were 4.3,3.5 and 1.4 %, respectively, all below 5.0 %, exhibitinggood precision.Analyses of ATP, ADP and AMP oflitchi fruit pericarp tissueThis HPLC method was applied to determine theconcentrations of ATP, ADP and AMP of pericarp tissuein litchi fruit at harvest. Fig. 2 shows the chromatogramsof ATP, ADP and AMP extracted from the litchi pericarptissue. There were 27.4 mg/g of ATP, 35.4 mg/g of ADPand 7.9 mg/g of AMP on a fresh mass (FM) basis. In thisstudy, 2 g of fresh litchi fruit pericarp tissue was enoughto determine the concentrations of ATP, ADP and AMP.Furthermore, the authors used this method to analyzethe concentrations of ATP, ADP and AMP of banana andlongan fruits, and found 10.9 mg/g FM of ATP, 12.2mg/g FM of ADP and 12.8 mg/g FM of AMP in bananafruit peel and 6.6 mg/g FM of ATP, 8.1 mg/g FM of ADPand 7.1 mg/g FM of AMP in longan fruit skin.In this study, the HPLC method could not separatethe peaks eluting between 2 and 3 min, whose ultravio-let absorption spectrum was at 254 nm (Fig. 2). Özogulet al. (14) reported that the unknown compounds in her-ring tissue could be the degradation products of ATP,such as inosine monophospate and hypoxanthine. How-ever, their identification in litchi fruit pericarp tissueneeds further investigation.ConclusionIn conclusion, an HPLC analysis of ATP, ADP andAMP concentrations in litchi fruit pericarp tissue wassimple and rapid to use. We suggest that the improvedmethod for identification and quantification of ATP,ADP and AMP should be a valuable tool in analyzing alarge number of fruit samples.AcknowledgementsFinancial support by the National Natural ScienceFoundation of China (No. 30425040 and 30430490) andthe International Foundation for Science (No. E2265-3) ishighly appreciated.References1. A. Pradet, P. Raymond, Adenine nucleotide ratios andadenylate energy charge in energy metabolism, Annu. Rev.Plant Physiol. 34 (1983) 199–224.2. R.J. Romani, S. Ozelkok, «Survival« of mitochondria in vi-tro: Physical and energy parameters, Plant Physiol. 51 (1973)702–707.3. K.C. Eastwell, P.K. Stumpf, Regulation of plant acetyl-CoAcarboxylase by adenylate nucleotides, Plant Physiol. 72 (1983)50–55.4. J.L. Harwood, Fatty acid metabolism, Annu. Rev. PlantPhysiol. Plant Mol. Biol. 39 (1988) 101–138.5. J. Ohlrogge, J. Browse, Lipid biosynthesis, Plant Cell, 7 (1995)957–970.6. V.N. Luzikov, Stabilization of the enzymic systems of theinner mitochondrial membrane and related problems, Sub-cell. Biochem. 2 (1973) 1–31.7. A.A. Saquet, J. Streif, F. Bangerth, Energy metabolism andmembrane lipid alterations in relation to brown heart de-velopment in ’Conference’ pears during delayed controlledatmosphere storage, Postharvest Biol. Technol. 30 (2003)123–132.8. D.J. Brown, H. Beevers, Fatty acid of rice coleoptiles in airand anoxia, Plant Physiol. 84 (1977) 555–559.9. A.A. Saquet, J. Streif, F. Bangerth, On the involvement ofadenine nucleotides in the development of brown heart in’Conference’ pears during delayed controlled atmospherestorage, Gartenbauwissenschaft, 66 (2001) 140–144.10. Y. Sugawara, M. Takeuchi, A simple and rapid method fordetermining cell survival in the cryopreserved shoot apex533H. LIU et al.: Determination of ATP, ADP and AMP in Litchi Fruit, Food Technol. Biotechnol. 44 (4) 531–534 (2006)Table 1. Comparative HPLC analyses of ATP, ADP and AMPCompound Meansa CV/%ATP 0.6111 1.80ADP 0.7667 1.28AMP 0.8663 1.30aData were expressed as peak areas (mm2) (N=6)Retention time/min0 1 2 3 4 5 6 70.0000.0050.0100.0150.0200.0250.030ATP ADP AMPA254nmFig. 2. HPLC chromatogram of ATP, ADP and AMP from litchifruit pericarp tissueusing luciferin-luciferase ATP assay, Plant Sci. 130 (1997)107–112.11. B. Naslund, L. Stahle, A. Lundin, B. Anderstam, P. Arner,J. Bergstrom, Luminometric single step urea assay usingATP-hydrolyzing urease, Clin. Chem. 44 (1998) 1964–1973.12. N.C. Yang, W.M. Ho, Y.H. Chen, M.L. Hu, A convenientone-step extraction of cellular ATP using boiling water forthe luciferin-luciferase assay of ATP, Anal. Biochem. 306(2002) 323–327.13. J.M. Ryder, Determination of adenosine triphosphate andits breakdown products in fish muscle by high-performanceliquid chromatography, J. Agric. Food Chem. 33 (1985) 678–680.14. F. Özogul, K.D.A. Taylor, P.C. Quantick, Y. Özogul, A ra-pid HPLC-determination of ATP-related compounds andits application to herring stored under modified atmo-sphere, Int. J. Food Sci. Technol. 35 (2000) 549–554.15. F.S. Anderson, R.C. Murphy, Isocratic separation of somepurine nucleotide, nucleoside and metabolites from biolo-gical extract by high-performance liquid chromatography,J. Chromatogr. 121 (1976) 251–262.16. M.T. Veciana-Nogues, M. Izquierdo-Pulido, M.C. Vidal-Ca-rou, Determination of ATP related compounds in fish andcanned tuna fish by HPLC, Food Chem. 59 (1997) 467–472.17. G. Manfredi, L.C. Yang, C.D. Gajewski, M. Mattiazzi, Mea-surements of ATP in mammalian cells, Methods, 26 (2002)317–326.18. J. Murray, A.B. Thomson, Reverse phase ion pair separa-tion of nucleotides and related products in fish muscle, J.High Resolut. Chromatogr. Chromatogr. Comunn. 6 (1983) 209–210.19. K. Samizo, R. Ishikawa, A. Nakamura, K. Kohama, A high-ly sensitive method for measurement of myosin ATPaseactivity by reversed-phase high-performance liquid chro-matography, Anal. Biochem. 293 (2001) 212–215.534 H. LIU et al.: Determination of ATP, ADP and AMP in Litchi Fruit, Food Technol. Biotechnol. 44 (4) 531–534 (2006)

A Simple and Rapid Determination of ATP, ADP and AMP Concentrations in Pericarp Tissue of Litchi Fruit by High Performance Liquid Chromatography

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FTB 44 (4) 531-534. Jednostavno i brzo određivanje masenog udjela ATP-a, ADP-a i AMP-a u perikarpu voća liči pomoću visokodjelotvorne tekućinske kromatografije  Sažetak  Razvijena je jednostavna i brza metoda određivanja masenog udjela adenozin trifosfata (ATP), adenozin difosfata (ADP) i adenozin monofosfata (AMP) visokodjelotvornom tekućinskom kromatografijom (HPLC) u perikarpu voća liči. Pri određivanju adenozin fosfata upotrijebljen je acetonitril koji skraćuje vrijeme postupka. Također je postignuta dobra ponovljivost (koeficijent varijacije iznosi 1,28-1,80 %) i obnovljivost (94,7-97,1 %). Koeficijenti korelacije s površinama pika u rasponu od 0 do 80 ng iznosili su 0,9946 za ATP; 0,9994 za ADP i 0,9974 za AMP. Ova je metoda primijenjena za određivanje masenog udjela adenozin fosfata u perikarpu voća liči pri berbi, pa je utvrđeno da je maseni udio ATP-a iznosio 27,4 µg/g, ADP-a 35,4 µg/g i AMP-a 7,9 µg/g, na bazi svježe tvari voća. 

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A simple and rapid method using high performance liquid chromatography (HPLC) was developed to determine levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) in litchi fruit pericarp tissue. This HPLC method used acetonitrile gradient elution and shortened the time required for determinations of adenosine phosphates. This analysis exhibited good repeatability (coefficients of variation 1.28–1.80 %) and recovery rate (94.7–97.1 %). The correlation coefficients of ATP, ADP and AMP with their peak areas at a range of 0–80 ng were 0.9946, 0.9994 and 0.9974, respectively. This method was applied to determine levels of adenosine phosphates in pericarp tissue of litchi fruit at harvest. There were 27.4 μg/g of ATP, 35.4 μg/g of ADP and 7.9 μg/g of AMP on a fresh mass basis

A Simple and Rapid Determination of ATP, ADP and AMP Concentrations in Pericarp Tissue of Litchi Fruit by High Performance Liquid Chromatography

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