Dante CS, Guibert EE, Rodríguez JV

Language: English
References: 26
Page: 140-46
PDF size: 236.86 Kb.

Text Extraction

We examined how different media composition of rewarming solutions affected ammonium detoxification function, urea synthesis and the viability of hepatocytes after 72 hs of cold storage in UW solution. Freshly isolated rat hepatocytes were incubated at 37 °C in a cell culture medium (MEM-E) with 3 mM glycine, 5 mM fructose and 2.5 mM adenosine (group 1) and in Krebs-Heinseleit buffer with 3 mM glycine, 5 mM fructose, 2 mM ornithine, 10 mM lactate and adenosine, that was used in two different concentrations: 2.5 mM (group 2) and 10 μM (group 3). We found that freshly isolated cells produced ammonium in group 1 and 2 but the cells were able to diminish ammonium extracellular concentration in group 3. Urea synthesis and ammonium extracellular concentration in group 1 was higher than in group 2. As a result of this observations, we used the Krebs-Heinseleit solution with addition of 10 μM adenosine to determinate the effect of hypothermic preservation on ammonium detoxification and urea synthesis ability of cells. In conclusion the addition of 2.5 mM adenosine into the rewarming medium interfered with the detection of ammonium detoxification of hepatic cells.

REFERENCES

1. Belzer FO and Southard JH. Principles of solid-organ preservation by cold storage. Transplantation 1988; 45: 673-676.

2. Sandker GW, Slooff MJ, Groothuis GM. Drug transport, viability and morphology of isolated rat hepatocytes preserved for 24 hs in University of Wisconsin solution. Biochemical Pharmacology 1992; 43: 1479-1485.

3. Southard JH, van Gulik TM, Ametani MS, Vreugdenhil PK, Lindell SL, Pienaar BL and Belzer FO. Important components of the UW solution. Transplantation 1990; 49: 251-257.

4. Rao KV, Norenberg MD. Cerebral energy metabolism in hepatic encephalopathy and hyperammonemia. Metabolic Brain Disease 2001; 16: 67-78.

5. Mamprin ME, Guibert EE, Rodríguez JV. Glutathione content during rinsing and rewarning process of rat hepatocytes preserved in University of Wisconsin solution. Cryobiology 2000; 40: 270-276.

6. Currin RT, Thurman RG, Lemaster JJ. Carolina rinse solution protects adenosine triphosphate-depleted hepatocytes against lethal cell injury. Transplantation Proceedings 1991; 23: 645-647.

7. Marsh DC, Belzer FO, Southard JH. Hypothermic preservation of hepatocytes. II Importance of Ca+2 and amino acids. Cryobiology 1990; 27: 1-8.

8. Marsh DC, Hjelmhaug PK, Vreugnhil JK, et al. Hypothermic preservation of hepatocytes. III Effects of resuspension media on viability after up to 7 days of storage. Hepatology 1991; 13: 500-506.

9. Spinelli SV, Rodríguez JV., Quintana AB, Mediavilla MG, Guibert EE. Engraftment and function of intrasplenically transplanted cold stored rat hepatocytes. Cell Transplantation 2002; 11: 2: 161-168.

10. Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biology 1976; 13: 30-83.

11. Rodriguez JV, Mamprin ME, Mediavilla MG and Guibert EE. Glutathione (GSH) movements during cold preservation of rat hepatocytes. Cryobiology 1998; 36: 236-244.

12. Mamprin ME, Rodriguez JV, Guibert EE. The importance of pH in resuspension media on viability of hepatocytes preserved in University of Wisconsin solution. Cell Transplantation 1995; 4: 269-274.

13. van Anken HC, Schiphorst ME. A kinetic determination of ammonium in plasma. Clinical Chimical Acta 1974; 56: 151-157.

14. Rahmatullah M, Boyde TR. Improvements in the determination of urea using diacetil monoxime; methods with and without deproteinization. Clinical Chimical Acta 1980; 107: 3-9.

15. Lund P, Cornell NW, Krebs HA. Effect of adenosine on the adenine nucleotide content and metabolism of hepatocytes. The Biochemical Journal 1975; 152: 593-599.

16. Cannon JR, Harvison, PJ, Rush GF. The effects of fructose on adenosine triphosphate depletion following mitochondrial dysfunction and lethal cell injury in isolated rat hepatocytes. Toxicology and Applied Pharmacology 1991; 108: 407-416.

17. Dickson RC, Bronk SF, Gores GJ. Glycine citoprotection during lethal hepatocellular injury from adenosine triphosphate depletion. Gastroenterology 1992; 102: 2098-2107.

18. Proelss HF, Wright BW. Rapid determination of ammonia in a perchloric acid supernate from blood, by use of a ammonia-specific electrode. Clinical Chemistry 1973; 19: 1162-1169.

19. Calligaris SD, Almada LL, Guibert EE, Tiribelli C, Rodriguez JV. Ammonium detoxifying activity is maintained after 72 hours of cold preservation of rat hepatocytes in University of Wisconsin (UW) solution. Crio-Letters 2002 (in press).

20. Briggs S, Freedland RA. Effect of ornithine and lactate on urea synthesis in isolated hepatocytes. The Biochemical Journal 1976; 160: 205-209.

21. Guinzberg R, Laguna I, Zentella A, Guzman R, Piña E. Effect of adenosine and inosine on ureagenesis in hepatocytes. The Biochemical Journal 1987; 245: 371-374.

22. Starke PE, Hock JB, Farber JL. Calcium-dependent and calcium-independent mechanisms of irreversible cell injury in cultured hepatocytes. Journal of Biological Chemistry 1986; 261: 3006-3012.

23. Vreugdenhil PK, Marsh DC, Belzer FO, Southard JH. Urea and protein synthesis in cold-preserved isolated rat hepatocytes. Hepatology 1992; 16: 241-246.

24. Baldwin SA, Mackey JR, Cass CE, Young JD. Nucleoside transporters: molecular biology and implications for therapeutic development. Molecular Medicine Today 1999; 5: 216-224.

25. Costell M, O’Connor JE, Miguez MP, Grisolia S. Effects of L-carnitine on urea synthesis following acute ammonia intoxication in mice. Biochem Biophys. Res Commun 1984; 120: 726-733.

26. Therrien G, Rose C, Butterworth J, Butterworth RF. Protective effect of L-carnitine in ammonia-precipitated encephalopathy in the portacaval shunted rat. Hepatology 1997; 25: 551-556.