Research Article: Comparative study between fructose 1-6 bisphosphate and histidine-tryptophan-ketoglutarate in liver preservation in rats submitted to total cold ischemia1

Date Published: July 08, 2020

Publisher: Sociedade Brasileira para o Desenvolvimento da Pesquisa em Cirurgia

Author(s): Fernanda Bombonato Smecellato, Lucas Ricardo Benfatti Marsilli, Julia Eico Nakamura, Maria Cecília Jordani, Paulo Roberto Barbosa Évora, Orlando Castro-e-Silva.


To compare Fructose-1,6-Bisphosphate (FBP) to Histidine-Tryptophan-Ketoglutarate (HTK) in liver preservation at cold ischemia.

Male rats (Sprague-Dawley: 280-340g) divided into three groups (n=7): Control; Fructose-1,6-bisphosphate (FBP); Histidine-Tryptophan-Ketoglutarate (HTK). Animals underwent laparotomy-thoracotomy for perfusion of livers with saline. Livers were removed and deposited into solutions. Mitochondria were isolated to determine State 3 (S3), State 4 (S4), Respiratory Control Ratio (RCR) and Swelling (S). Liver enzymes (AST, ALT, LDH) were determined in solution. At tissue, Malondialdehyde (MDA) and Nitrate (NOx) were determined. All parameters were analyzed at 0.6 and 24 hours of hypothermic preservation. Statistics analysis were made by Mann-Whitney test (p<0.05). Regarding ALT, there was a difference between FBP-6h/HTK-6h, lower in HTK. Regarding AST, there was a significant difference between FBP-24h/HTK-24h, lower in FBP. Regarding NOx, there was a difference between 0h and 6h, as well as 0h and 24h for both solutions. Regarding S3, there was a significant difference in 24h compared to Control-0h for both solutions, and a significant difference between FBP-6h/FBP-24h. Regarding S4, there was a difference between Control-0h/HTK-24h and FBP-24h/HTK-24h, higher in HTK. There was a difference between Control-0h/FBP-24h for Swelling, higher in FBP. Fructose-1,6-Bisphosphate showed better performance at nitrate and aspartate aminotransferase compared to histidine-tryptophan-ketoglutarate.

Partial Text

Liver transplantation is used in situations in which patients with liver disease have a life expectancy of less than 20% at the end of 12 months, if they are not transplanted, and in those in which the risk of mortality exceeds the rate resulting from the transplant itself. It is considered a form of treatment for patients with terminal liver failure due to chronic cholestatic and hepatocellular liver diseases, metabolic and vascular liver diseases, primary liver tumor and trauma1,2. More than 10.000 liver transplants are performed worldwide per year3. This high number is certainly a reflection of innumerable research carried out with the aim of improving the conditions of the transplant. Among these researches, are those involved with the improvement of preservation solutions for liver. With this, it is possible to transport organs over great distances and, consequently, use the vast majority of organs available for donation1.

The Animal Experimentation Ethics Committee approved the study of the Faculdade de Medicina de Ribeirão Preto of Universidade de São Paulo (FMRP-USP). The rats were kept under controlled light conditions (12 hours of light and 12 hours of darkness), temperature 23ºC, relative humidity 55%, and free access to water and food for rodents.

The results obtained in the serum determinations of ALT, AST, and LDH are shown in Figure 1 A-C, respectively. The two solutions showed significant differences between studied times (0, 6, and 24) for ALT, AST, and LDH, with increased levels over time. Regarding ALT, there was a difference between the FBP 6h x HTK 6h groups, with lower rates of the enzyme for HTK at that time. Regarding AST, there was a significant difference between the FBP 24h x HTK 24 groups, with much lower levels of the enzyme for FBP, showing a beneficial effect of FBP about HTK at that time. Regarding LDH, there were similar results for both solutions, with no significant differences between them at the same time and shows the same ability of FBP concerning HTK in preserving the liver for this enzyme.

Some authors suggest that FBP crosses cell membranes to restore depressed glycolytic activity, not only as a metabolic regulator, but also as a metabolic substrate for glycolysis while maintaining intracellular ATP14. This can be proven using carbon 13 nuclear magnetic resonance, such that the FBP marked with exogenous carbon 13 can visibly cross the cell membrane14-17. It is known that changes in cell membranes during the ischemia process lead to the flow ionic and consequent increase in intracellular Ca2 levels. FBP can interact with the biomembrane, modifying ionic permeability, in order to maintain its stability. Thus, FBP has a Ca2 chelating action, inhibiting the increase in intracellular Ca2 concentration in Kupffer cells isolated from rats18. FBP also has anti-inflammatory action, since it is responsible for inhibiting the proliferation of activated mononuclear cells by mitogen, mainly helper T cells, which are most affected. This is due to the FBP’s ability to inhibit IL-2 expression19.

The present study showed better protective effects of the FBP, at NOx and AST, compared to HTK. These data are suggestive of a protective effect on hepatocyte damage and less free radical production with consequent better endothelial function. Since the FBP presented some similar and other superior results, in addition to the economic advantage when compared to the high cost of HTK, these results indicate FBP as a viable solution in the preservation of the liver graft in cold ischemia.




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