A novel method for determination of ATP, ADP, and AMP contents of a single pancreatic islet before transplantation

Abstract

Assessment of islet viability before transplantation is mandatory for successful clinical transplantation. ATP content or energy charge (EC) of islets may represent a good parameter to assess viability. We have introduced a novel bioluminescent enzymatic cycling assay using synthetic firefly luciferase, pyruvate kinase) (PK), and pyruvate orthophosphate dikinase (PPDK) to determine adenine nucleotides (AN) in isolated islets. A complete assay requires several minutes. The ATP contents of 1, 3, 10, 30 or 100 islets each with a diameter of 150 microm were 9.95 +/- 0.03, 28.3 +/- 1.18, 89.5 +/- 1.48, 282 +/- 10.2, and 673 +/- 27.1 pmol, respectively, showing a relatively constant ATP content per 30 islets (9.95, 9.42, 8.95, and 9.41 pmol/IEQ). ECs of each group were 0.74 +/- 0.02, 0.74 +/- 0.02, 0.75 +/- 0.02, 0.74 +/- 0.02, and 0.65 +/- 0.01, respectively, a value that was quite constant up to 30 islets. The quantity of ATP and EC in a single islet can be measured quickly and reproducibly, offering a new method to determine viability of isolated islets prior to transplantation.