- Email: [email protected]
Evaluation of Energy State of Islet Independent of Size Using a Newly Developed ATP Bioluminescence Assay
Evaluation of Energy State of Islet Independent of Size Using a Newly Developed ATP Bioluminescence Assay S. Ishii, T. Saito, K. Ise, Y. Sato, T. Tsutiya, A. Kenjo, T. Kimura, T. Anazawa, S. Suzuki, M. Terashima, and M. Gotoh ABSTRACT ATP content or energy charge (EC) of islets may be a good parameter to assess viability. In this study, we examined adenine nucleotides and EC of freshly isolated or 24-hour cultured rat islets of various diameters using a novel bioluminescent enzymatic cycling assay system. For freshly isolated islets, ATP content and islet diameter showed a high correlation (r ⫽ 0.842, P ⬍ .001), but a significant correlation was not observed for cultured islets (r ⫽ 0.284) when all islets were included for the analysis. When only the cultured islets with a diameter ⬍350 m were included for analysis, a significant correlation was observed (r ⫽ 0.719). EC of freshly isolated islets fluctuated widely irrespective of diameter, in contrast with results of 24-hour cultured islets, which showed a stable, high EC, regardless of diameter. These data suggest that the ATP content of islets correlates with the islet size and that EC of islets widely fluctuates following isolation, indicating a significant role of monitoring ATP and EC of islets before transplantation.
R
APID ASSESSMENT of islet viability before transplantation is mandatory for successful clinical islet transplantation. ATP content or energy charge (EC) may be a good parameter to assess islet viability. We have shown that a bioluminescent enzymatic cycling assay using synthetic firefly luciferase, pyruvate kinase (PK) and pyruvate orthophosphate dikinase (PPDK) reflects the adenine nucleotides (AN) of a single islet of 150 m diameter within several minutes.1 In this study, we examined AN and EC of freshly isolated or 24-hour cultured islets of various diameters.
ATP Bioluminescence Assay AN were measured as previously described,1,3 using newly developed bioluminescence reagents containing firefly luciferase supplied by Kikkoman Corp.
Statistical Analysis Correlation between islet diameter and AN or EC was examined for statistical significance using StatView 4.5 statistical software. P values of ⬍.05 denoted the presence of a significant statistical difference.
RESULTS AND DISCUSSION METHODS Islet Isolation and Culture Pancreatic islets were isolated from 7- to 8-week-old male WS rats by stationary collagenase digestion followed by Ficoll discontinuous gradients as described previously.2 Handpicked freshly isolated islets were placed in 96-well flat bottom microplates (BD bioscience, San Jose, Calif, USA) with 200 L of RPMI 1640. For cultured islet experiments, freshly isolated islets were cultured in a Petri dish (90 mm in diameter, Sumitomo bakelite Co. Ltd, Tokyo, Japan) with RPMI 1640 for 24 hours at 37°C in 5% carbon dioxide 95% air in a humidified atmosphere. The diameter of the handpicked islets was measured by photomicrograph under an inverted microscope with a scale. For AN measurement, 20 L of 0.5 N HClO4 was added to each well, before the liquid was aspirated and frozen at ⫺80°C until assay.
ATP content was compared with the diameter of freshly isolated or 24-hour cultured islets (Fig 1). For freshly isolated islets, ATP content and islet diameter showed a highly significant correlation (r ⫽ 0.842, P ⬍ .001). A similar From the Department of Surgery, Fukushima Medical University, Fukushima, Japan. Supported in part by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology and in part by Grant-in-Aid for Research on Human Genome, Tissue Engineering Food Biotechnology, Health Sciences Research Grants, Ministry of Health, Labour and Welfare of Japan. Address reprint requests to Mitsukazu Gotoh, MD, Department of Surgery I, Fukushima Medical University, I Hikarigaoka, Fukushima 960-1295, Japan. E-mail: [email protected]
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.09.134
Transplantation Proceedings, 37, 3499 –3500 (2005)
3499
3500
correlation was observed when analysis used the second or third power of diameter instead of diameter. On the other hand, for cultured islets, no significant correlation was found when all islets were included for the analysis (r ⫽ 0.284). The cultured islets with a diameter ⬎350 m showed relatively constant ATP levels, resulting in no correlation (r ⫽ 0.017). When only the cultured islets with a diameter ⬍350 m were included for analysis, a significant correlation was observed (r ⫽ 0.719). These results indicate that 24-hour culture causes some damage to relatively large islets. EC was compared between freshly isolated and 24-hour cultured islets (Fig 2). EC of freshly isolated islets fluctuated widely irrespective of a diameter, which contrasted with EC of 24-hour cultured islets that were stable high EC values regardless of diameter. These results suggest that islets with low EC might restore EC or die during the culture. During culture, some freshly isolated islets were found to be dispersed, sometimes leaving no cells. Large islets usually experience central necrosis in a standard
ISHII, SAITO, ISE ET AL
Fig 2. Correlation between EC and the diameter of freshly isolated islet (Œ) and 24-hour cultured islet (‘). EC of freshly isolated islets fluctuated widely, which is contrasted with those of 24-hour cultured islets showing high EC regardless of a diameter.
culture system.4 In the line of these findings together, it is reasonable to assume that ATP content of islets is highly correlated with a viable size of islet and EC of islet is fluctuating during isolation and culture procedure. Again, these data indicate a significance of bioluminescent enzymatic cycling assay system for functional studies of islets.
REFERENCES
Fig 1. Correlation between ATP and the diameter of freshly isolated islet (Œ) and 24-hour cultured islet (‘). High correlation was observed for freshly isolated islet (r ⫽ 0.842). High correlation was observed for 24-hour cultured islets when islets with a diameter ⬎350 m were excluded (r ⫽ 0.719).
1. Ishii S, Sato Y, Terashima M, et al: A novel method for determination of ATP, ADP, and AMP contents of a single pancreatic islet before transplantation. Transplant Proc 36:1191, 2004 2. Gotoh M, Maki T, Kiyoizumi T, Satomi S, et al: An improved method for isolation of mouse pancreatic islets. Transplantation 40:437, 1985 3. Hattori N, Sakakibara T, Kajiyama N, et al: Enhanced microbial biomass assay using mutant luciferase resistant to benzalkonium chloride. Anal Biochem 319:287, 2003 4. Ono J, Lacy PE, Michael HE, et al: Studies of the functional and morphologic status of islets maintained at 24°C for four weeks in vitro. Am J Pathol 97:489, 1979
R
APID ASSESSMENT of islet viability before transplantation is mandatory for successful clinical islet transplantation. ATP content or energy charge (EC) may be a good parameter to assess islet viability. We have shown that a bioluminescent enzymatic cycling assay using synthetic firefly luciferase, pyruvate kinase (PK) and pyruvate orthophosphate dikinase (PPDK) reflects the adenine nucleotides (AN) of a single islet of 150 m diameter within several minutes.1 In this study, we examined AN and EC of freshly isolated or 24-hour cultured islets of various diameters.
ATP Bioluminescence Assay AN were measured as previously described,1,3 using newly developed bioluminescence reagents containing firefly luciferase supplied by Kikkoman Corp.
Statistical Analysis Correlation between islet diameter and AN or EC was examined for statistical significance using StatView 4.5 statistical software. P values of ⬍.05 denoted the presence of a significant statistical difference.
RESULTS AND DISCUSSION METHODS Islet Isolation and Culture Pancreatic islets were isolated from 7- to 8-week-old male WS rats by stationary collagenase digestion followed by Ficoll discontinuous gradients as described previously.2 Handpicked freshly isolated islets were placed in 96-well flat bottom microplates (BD bioscience, San Jose, Calif, USA) with 200 L of RPMI 1640. For cultured islet experiments, freshly isolated islets were cultured in a Petri dish (90 mm in diameter, Sumitomo bakelite Co. Ltd, Tokyo, Japan) with RPMI 1640 for 24 hours at 37°C in 5% carbon dioxide 95% air in a humidified atmosphere. The diameter of the handpicked islets was measured by photomicrograph under an inverted microscope with a scale. For AN measurement, 20 L of 0.5 N HClO4 was added to each well, before the liquid was aspirated and frozen at ⫺80°C until assay.
ATP content was compared with the diameter of freshly isolated or 24-hour cultured islets (Fig 1). For freshly isolated islets, ATP content and islet diameter showed a highly significant correlation (r ⫽ 0.842, P ⬍ .001). A similar From the Department of Surgery, Fukushima Medical University, Fukushima, Japan. Supported in part by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology and in part by Grant-in-Aid for Research on Human Genome, Tissue Engineering Food Biotechnology, Health Sciences Research Grants, Ministry of Health, Labour and Welfare of Japan. Address reprint requests to Mitsukazu Gotoh, MD, Department of Surgery I, Fukushima Medical University, I Hikarigaoka, Fukushima 960-1295, Japan. E-mail: [email protected]
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.09.134
Transplantation Proceedings, 37, 3499 –3500 (2005)
3499
3500
correlation was observed when analysis used the second or third power of diameter instead of diameter. On the other hand, for cultured islets, no significant correlation was found when all islets were included for the analysis (r ⫽ 0.284). The cultured islets with a diameter ⬎350 m showed relatively constant ATP levels, resulting in no correlation (r ⫽ 0.017). When only the cultured islets with a diameter ⬍350 m were included for analysis, a significant correlation was observed (r ⫽ 0.719). These results indicate that 24-hour culture causes some damage to relatively large islets. EC was compared between freshly isolated and 24-hour cultured islets (Fig 2). EC of freshly isolated islets fluctuated widely irrespective of a diameter, which contrasted with EC of 24-hour cultured islets that were stable high EC values regardless of diameter. These results suggest that islets with low EC might restore EC or die during the culture. During culture, some freshly isolated islets were found to be dispersed, sometimes leaving no cells. Large islets usually experience central necrosis in a standard
ISHII, SAITO, ISE ET AL
Fig 2. Correlation between EC and the diameter of freshly isolated islet (Œ) and 24-hour cultured islet (‘). EC of freshly isolated islets fluctuated widely, which is contrasted with those of 24-hour cultured islets showing high EC regardless of a diameter.
culture system.4 In the line of these findings together, it is reasonable to assume that ATP content of islets is highly correlated with a viable size of islet and EC of islet is fluctuating during isolation and culture procedure. Again, these data indicate a significance of bioluminescent enzymatic cycling assay system for functional studies of islets.
REFERENCES
Fig 1. Correlation between ATP and the diameter of freshly isolated islet (Œ) and 24-hour cultured islet (‘). High correlation was observed for freshly isolated islet (r ⫽ 0.842). High correlation was observed for 24-hour cultured islets when islets with a diameter ⬎350 m were excluded (r ⫽ 0.719).
1. Ishii S, Sato Y, Terashima M, et al: A novel method for determination of ATP, ADP, and AMP contents of a single pancreatic islet before transplantation. Transplant Proc 36:1191, 2004 2. Gotoh M, Maki T, Kiyoizumi T, Satomi S, et al: An improved method for isolation of mouse pancreatic islets. Transplantation 40:437, 1985 3. Hattori N, Sakakibara T, Kajiyama N, et al: Enhanced microbial biomass assay using mutant luciferase resistant to benzalkonium chloride. Anal Biochem 319:287, 2003 4. Ono J, Lacy PE, Michael HE, et al: Studies of the functional and morphologic status of islets maintained at 24°C for four weeks in vitro. Am J Pathol 97:489, 1979