ABSTRACT
The firefly luciferase assay of adenosine-5'-triphosphate (ATP) is used in a simple procedure for the determination of adenine nucleotides in whole blood and red blood cells. Cells are lysed with cold trichloroacetic acid and nucleotides are determined in the lysate after dilution with buffer. In a comparative study with the spectrophotometric assay of ATP, extracts were prepared by centrifugation of the lysate to remove interfering turbidity. This step resulted in a significant loss of ATP in the clear supernatant. This loss could be avoided by determination of ATP by the firefly luciferase assay in diluted uncentrifuged lysates. The yields of ATP, ATP + ADP and ATP + ADP + AMP were compared in a red blood cell preparation after lysis with trichloroacetic acid and another lytic method based on the detergent triton X-100. The precision was better with acid whereas yields were essentially the same with both methods. The method of standardization, external or internal, was of little practical importance regarding yields and precision, but internal calibration is recommended to ensure that there is no interaction of the luciferase reaction with lytic reagent or sample.
Subject(s)
Adenosine Diphosphate/blood , Adenosine Monophosphate/blood , Adenosine Triphosphate/blood , Luciferases/blood , Spectrophotometry/methods , Calibration , Erythrocytes/metabolism , Humans , Indicators and Reagents , Luminescent MeasurementsABSTRACT
The bioluminescent firefly luciferase assay for ATP was used to measure adenylate kinase activity in plasma. The formation of ATP from ADP was measured continuously in a coupled assay using a luminometer. Optimal analytical conditions were determined for the coupled reaction. The assay was used to follow accumulation of adenylate kinase in plasma of different preparations of stored red blood cells. Adenylate kinase was found to be released concomitantly with hemoglobin during aging. There was a high degree of correlation between the amount of accumulated hemoglobin and adenylate kinase. The assay was also used to measure lysis of stored platelets during aging.
Subject(s)
Adenylate Kinase/blood , Erythrocytes/enzymology , Phosphotransferases/blood , Adenosine Diphosphate/pharmacology , Animals , Blood Preservation , Coleoptera , Humans , Kinetics , Luciferases , Luminescent MeasurementsABSTRACT
The levels of ATP and total adenine nucleotides (ATP + ADP + AMP) were determined by firefly luciferase assay in red blood cells during storage for 5 weeks at 4 degrees C. With few exceptions, no significant differences in nucleotide levels were found between whole blood stored in CPD-adenine and various preparations of red blood cells in CPD-adenine or CPD with saline-adenine-glucose (SAG) as additive. The levels of ATP and total adenine nucleotides during storage are discussed in relation to glucose levels, extracellular pH and shelf life of the red blood cells.
