The ATP/DNA Ratio Is a Better Indicator of Islet Cell Viability Than the ADP/ATP Ratio

Abstract

Real-time, accurate assessment of islet viability is critical for avoiding transplantation of nontherapeutic preparations. Measurements of the intracellular ADP/ATP ratio have been recently proposed as useful prospective estimates of islet cell viability and potency. However, dead cells may be rapidly depleted of both ATP and ADP, which would render the ratio incapable of accounting for dead cells. Since the DNA of dead cells is expected to remain stable over prolonged periods of time (days), we hypothesized that use of the ATP/DNA ratio would take into account dead cells and may be a better indicator of islet cell viability than the ADP/ATP ratio. We tested this hypothesis using mixtures of healthy and lethally heat-treated (HT) rat insulinoma cells and human islets. Measurements of ATP/DNA and ADP/ATP from the known mixtures of healthy and HT cells and islets were used to evaluate how well these parameters correlated with viability. The results indicated that ATP and ADP were rapidly (within 1 hour) depleted in HT cells. The fraction of HT cells in a mixture correlated linearly with the ATP/DNA ratio, whereas the ADP/ADP ratio was highly scattered, remaining effectively unchanged. Despite similar limitations in both ADP/ADP and ATP/ DNA ratios, in that ATP levels may fluctuate significantly and reversibly with metabolic stress, the results indicated that ATP/DNA was a better measure of islet viability than the ADP/ATP ratio.Islet cell transplantation is emerging as a promising therapy for the treatment of type 1 diabetes. [1][2][3][4][5] Despite recent advances, the transplantation of islets poses a unique challenge with respect to achieving a consistent clinical outcome. Part of this challenge is being able to reliably and rapidly assess clinical islet quality through the quantification of viability and function before transplantation. Current viability assays are limited in their ability to accurately predict transplantation outcomes in vivo. 6 Consequently, to improve the clinical islet transplantation outcomes, it is imperative to develop more accurate viability assays.A proposed method to assess islet viability and potency before transplantation is quantification of the ADP/ATP ratio. 6 -10 This ratio has been specifically applied in discriminating islet preparations that are suitable for clinical transplantation from those that are not. 6 However, this application may be problematic under certain conditions. Intracellular ADP and ATP levels fluctuate rapidly because these high-energy phosphate molecules are rapidly produced and consumed in many intracellular biochemical reactions. It is widely known that viable cells turn over entire ATP stores on the order of minutes, and that dead cells are incapable of replenishing To better account for dead cells and to more accurately assess the viability of an islet preparation, we suggest the use of an ATP/DNA ratio. DNA does not degrade as rapidly as ADP or ATP in a dead cell. Therefore, using direct measurements of DNA, dead cells in an islet preparation...

Development of Poly(Ethylene Glycol) Hydrogels for Salivary Gland Tissue Engineering Applications

“…1B). Cellular ATP has a rapid turnover (t 1/2 * minutes), and dead cells experience a rapid drop in ATP levels, [72][73][74] making ATP an appropriate surrogate analyte for viability. 39 Immediately after exposure to chain-addition methacrylate polymerization conditions, SMG cells experienced a significant decrease in viability, suggesting acute cell death (Fig.…”

Quantitative Assessment of Islets of Langerhans Encapsulated in Alginate

“…15,16 Examination of viability by assessment of membrane integrity with vital stains such as fluorescein diacetate/propidium iodide does not reflect true viability because it may not account for cells undergoing early cell death processes, during which cells have not yet developed damage to their cell membrane. 8,9,17,18 Measures of cellular energetic state, including ATP content [19][20][21][22][23][24] and ATP/ADP and related ratios, [25][26][27] have been investigated with modest success. ATP content varies enormously between islets from different species, between human islet preparations, [20][21][22] with time in culture, 20,21 and with donor age.…”

Metabolic Profile of Pancreatic Acinar and Islet Tissue in Culture

“…Aliquots of acinar and islet tissue were taken on different days of culture and assessed for DNA content using a commercially-available spectrophotometric assay (Quant-IT PicoGreen dsDNA kit, Invitrogen, Grand Island, NY) and a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA)(10). The quantity of DNA in an aliquot was extrapolated to the entire preparation in culture based on volume.…”

“…The quantity of DNA in an aliquot was extrapolated to the entire preparation in culture based on volume. DNA content measured on Days 6-9 were normalized to the DNA content measured on Day 0 to yield a “ DNA recovery ”, which represented a “total tissue” recovery, since DNA is stable even in dying tissue (10). DNA recoveries measured on Days 6-9 were then multiplied by the OCR/DNA (fractional viability) to yield an “ OCR recovery ”, which represented the “viable tissue” recovery in the preparation.…”