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Adenine Nucleotide Assays and Issues Print E-mail

Adenine Nucleotide Assays by HPLC and some issues

  An excerpt from “Mitochondria Interest Group“ ( ) mail list.
2013-2014

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Q: I am interested in quantifying the amount of AMP / ADP / ATP I would like to quantify these ratios within the same piece of tissue.

A1: (modified from the post of Dr.Sukanta Jash):
  I have measured AMP/ADP/ATP ratio in rat skeletal muscle injury sample with the help of reverse phase HPLC method through modification with the Stocchi et al (1985) protocol. It gives very reproducible result. Below is my modified protocol.

 Preparation of muscle extracts for nucleotide estimation. 50 mg of Muscle tissues were taken on a cold plate (cooled at liquid nitrogen).The tissues were crushed into dust in liquid nitrogen and transferred to a 1.5-mL tube, to which 500 μl  of ice-cold 70% perchloric acid/g (wet wt) was added and kept for 5 minutes on ice. The homogenates were centrifuged at 1500 x g at 4°C for 20 min. The supernatant were neutralized with 14 μl of 0.5 M triethanolamine with 2.0 M K2CO3, added to 100 μl of the supernatant, kept on ice for 10 min, and centrifuged at 1500x g at 4°C for 20 min.

 HPLC assay: To determine ATP, ADP and AMP, HPLC was performed using a Waters 515 HPLC Pump and Waters 2998 photodiode-array detector, controlled with Waters Pump Control Module II. Data analyses were performed with Empower 2 software. HPLC was run as in Stocchi et al (1985). An aliquot (100 mL) of each sample was passed through an XTerra RP18, 5 μm, (4.6 × 250 mm i.d.) column. Detection of absorbance occurred at 257 nm, and the flow rate was set at 1 mL/min. A gradient was initiated using 2 buffers, in which buffer A consisted of100 mM KH2PO4, pH 6, and buffer B was 10% (v:v) acetonitrile, 100 mM KH2PO4, pH 6. The gradient was changed from 100% buffer A, 9 min; 0-25% of buffer B, 6 min; increase to 90% buffer B, 2.5 min; and up to 100% of buffer B, 2 min, held for 6 min. Peaks were identified from their retention times and by chromatography of standards. To determine ATP, ADP and AMP, HPLC was performed using a Waters 515 HPLC Pump and Waters 2998 photodiode-array detector, controlled with Waters Pump Control Module II. Data analyses were performed with Empower 2 software. HPLC was run as in Stocchi et al (1985) An aliquot (100 mL) of each sample was passed through an XTerra RP18, 5 μm, (4.6 × 250 mm i.d.) column. Detection of absorbance occurred at 257 nm, and the flow rate was set at 1 mL/min. A gradient was initiated using 2 buffers, in which buffer A consisted of100 mM KH2PO4, pH 6, and buffer B was 10% (v:v) acetonitrile, 100 mM KH2PO4, pH 6. The gradient was changed from 100% buffer A, 9 min; 0-25% of buffer B, 6 min; increase to 90% buffer B, 2.5 min; and up to 100% of buffer B, 2 min, held for 6 min. Peaks were identified from their retention times and by chromatography of standards.

A2: (modified from the post of Dr. William A. Irwin):
  The concern with the reverse phase HPLC method is that for the most important analytes ADP and ATP the peaks often merge, with differences in retention times of less than 1 minute. The anion HPLC method better separates ATP and ADP by about 4 minutes in the retention times and the run time is about twice as fast.

 I utilized anion exchange high performance liquid chromatography (HPLC) to separate and simultaneously quantitate ATP, ADP, AMP and adenine levels in the same sample aliquot, so that cellular energy charge can be calculated. It worked very well and didn't have the several artifacts of the luciferase-based assays. The reproducibility of this assay for nucleotide standards was excellent, typically +/- 1.0 % of the peak area. The correlation between nucleotide quantity and peak area for me was 0.998. The anion exchange method has considerably better separation of adenine nucleotides than a reverse phase column method and is the method that Sigma has employed to assay its adenine nucleotides.

 Adenine Nucleotide Assay: A strongly basic anion column (i.e. Dionex ProPac PA1 or equivalent) with a 4x250 mm primary column and a 4x50 mm guard column was utilized and adenine nucleotides were monitored by absorbance at 259 nm. The mobile phase buffer contained 50 mM sodium acetate at pH=4.5 and nucleotide elution was obtained by linearly increasing the concentration of sodium chloride from 0 to 400 mM in this buffer over a period of 16 minutes. The retention times for adenosine, AMP, ADP, and ATP for our equipment were 4.4, 7.8, 11.3 and 15.0 minutes, respectively when using a flow rate of 1.0 ml/minute. The detection of <0.25 nanomol per peak is easily measured with this assay.

 The tissue or cell extract is homogenized on ice and samples are de-proteinized by making cell extracts 1 M in perchloric acid using ice-cold 3 M perchloric acid, then centrifuged. An aliquot of this supernatant is brought to neutral pH with ice-cold 9 M potassium hydroxide and centrifuged. This supernatant is filtered with a 0.45 micron syringe filter before injection into the HPLC. Prepared samples are stored on ice or frozen for short periods until analyzed.

 The anion chromatography adenine nucleotide method was published by Kato, Y, Nakamura, K, Hashimoto, T, (1982) J Chromatography 245: 193-211 and Kamiike, W et al (1982) J Biochem 91: 1349.

Additional comments (modified from the post of Dr. Christos Chinopoulos):
  Note that a great part of the adenine nucleotides is bound to proteins [13], a notion supported by the fact that rat liver mitochondria retain more than 50% of their total adenine nucleotide content after permeabilization by toluene [14]. Because of this potential binding of adenine nucleotides to proteins [1;6;11;20] the relationship between the measured total ATP/ADP ratio to free intramitochondrial ATP/ADP ratio is difficult to predict. For example, previous data by Vignais show that a large fraction (75-80%) of the ATP produced by phosphorylation of added ADP within the inner mitochondrial membrane is released into the matrix space before being transported out from the mitochondria; only a small part (20-25%) is released directly outside the mitochondria without penetrating the matrix space [27]. It is therefore inferred that there are separate intramitochondrial pools of adenine nucleotides, one near the Adenine nucleotide translocase (ANT) – ATPase complex, and another pool being located in the “bulk” of the matrix volume. The notion of matrix microcompartmentation of adenine nucleotides emanated from several laboratories [5;16;17;26;27], but is not yet accepted unequivocally in the field [7;10;12].

[1] P.D. Boyer, Toward an adequate scheme for the ATP synthase catalysis. Biochemistry (Mosc.) 66 (2001) 1058-1066.
[5] H.C. Hamman, R.C. Haynes, Jr., Elevated intramitochondrial adenine nucleotides and mitochondrial function. Arch.Biochem.Biophys. 223 (1983) 85-94.
[6] D.A. Harris, J. Rosing, R.J. van de Stadt, E.C. Slater, Tight binding of adenine nucleotides to beef-heart mitochondrial ATPase. Biochim.Biophys.Acta 314 (1973) 149-153.
[7] K.J. Hartung, G. Bohme, W. Kunz, Involvement of intramitochondrial adenine nucleotides and inorganic phosphate in oxidative phosphorylation of extramitochondrially added adenosine-5'-diphosphate. Biomed.Biochim.Acta 42 (1983) 15-26.
[10] H.W. Heldt, E. Pfaff, Adenine nucleotide translocation in mitochondria. Quantitative evaluation of the correlation between the phosphorylation of endogenous and exogenous ADP in mitochondria. Eur.J.Biochem. 10 (1969) 494-500.
[11] J.M. Jault, W.S. Allison, Hysteretic inhibition of the bovine heart mitochondrial F1-ATPase is due to saturation of noncatalytic sites with ADP which blocks activation of the enzyme by ATP. J.Biol.Chem. 269 (1994) 319-325.
[12] G. Letko, U. Kuster, J. Duszynski, W. Kunz, Investigation of the dependence of the intramitochondrial [ATP]/[ADP] ratio on the respiration rate. Biochim.Biophys.Acta 593 (1980) 196-203.
[13] C.J. Lusty, Carbamoylphosphate synthetase I of rat-liver mitochondria. Purification, properties, and polypeptide molecular weight. Eur.J.Biochem. 85 (1978) 373-383.
[14] M.A. Matlib, W.A. Shannon, Jr., P.A. Srere, Measurement of matrix enzyme activity in isolated mitochondria made permeable with toluene. Arch.Biochem.Biophys. 178 (1977) 396-407. [16] M.S. Murthy, S.V. Pande, Microcompartmentation of transported carnitine, acetylcarnitine and ADP occurs in the mitochondrial matrix. Implications for transport measurements and metabolism. Biochem.J. 230 (1985) 657-663. [17] T.A. Out, E. Valeton, A. Kemp, Jr., Role of the intramitochondrial adenine nucleotides as intermediates in the uncoupler-induced hydrolysis of extramitochondrial ATP. Biochim.Biophys.Acta 440 (1976) 697-710. [20] A.E. Senior, S. Nadanaciva, J. Weber, Rate acceleration of ATP hydrolysis by F(1)F(o)-ATP synthase. J.Exp.Biol. 203 (2000) 35-40. [26] P.V. Vignais, Molecular and physiological aspects of adenine nucleotide transport in mitochondria. Biochim.Biophys.Acta 456 (1976) 1-38. [27] P.V. Vignais, P.M. Vignais, J. Doussiere, Functional relationship between the ADP/ATP-carrier and the F1-ATPase in mitochondria. Biochim.Biophys.Acta 376 (1975) 219-230.

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