Adenine nucleotide depletion in cryopreserved human cardiac valves: The “stunned” leaflet interstitial cell population

Preparation protocols for human cardiac valves are intended to minimize cytotoxicity because it has been thought that viable leaflet interstitial cells may enhance homograft durability. Preimplantation factors influencing the status of these cells at the time of transplantation include ischemia, disinfection, and cryopreservation freezing programs. In these experiments, adenine nucleotide quantitation was undertaken to assess metabolic consequences of preparation; preharvest ischemia served as an independent variable to examine the relationship between time of procurement (postmortem) and high-energy phosphate status of the cryopreserved leaflets at thaw. Nucleotides were measured using high-performance liquid chromatography performed on extracts of semilunar cusps from 25 cryopreserved human valves with documented ischemic times. Results indicate total adenine nucleotides (TAN; [ATP] + [ADP] + [AMP], in nmol TAN/mg leaflet protein) are higher (P < 0.05) after <2 h of harvest ischemia (1.16 ± 0.36) than with ischemic times of 3-6 h (undetected), 7-12 h (0.18 ± 0.07), and 13-20 h (0.06 ± 0.06). Depletion of ATP was similar, with many leaflets devoid of detectable levels. Net utilization of leaflet energy stores demonstrates time dependency when assayed after completed processing. However, relatively elevated catabolites, even with brief ischemia, and infrequently identified ATP, ADP, and AMP, suggest a consumption so accelerated that the following cryopreservation it is virtually independent of procurement-associated ischemia. We conclude resumption of a functional cell population obligates significant de novo phosphoanhydride boned reformation or a repopulation of dead/dying interstitial cells from a subset surviving the apparently severe rigors of valve preparation.