Comparison Of Functions By Liver-Derived Proteins Between Banna Minipig Inbred Line (BMI) And Human

The discrepancy between the demand and supply of organs for clinical transplantation remains a major problem. Xenotransplantation is considered a promising possible solution to the problem. The pig is considered the primary alternative species because of ethical considerations, breeding characteristics, and its compatible size. Recently people have made a great progress in the area. For all of the advantages of xenotransplantation, however, there are also daunting hundles to success. These hurdles include the immune response of the recipient against the transplant, the potential limitation and the physiologic function of the transplant in a foreign host, and the possibility that novel infectious organisms might be transmitted from the transplant to the recipient. However, the risk of PERV transmission (porcine endogenous retrovirus) remains, and is a preoccupying issue. Nevertheless, no documented case of this particular risk has yet been recorded. Severalimmunologic barriers could be overcome in the future by the induction of a state of tolerance or transgenic pigs. However, comparability of liver physiologic function between donor animals and human, the essential premise for successful xenotransplantation, had not been clearly studied. It is not sufficiently investigated whether under such physiological and anatomic differences a long-term survival of xenograft or recipient is possible. Are they able to maintain the metabolism in a foreign organ or body and for how long? As the unique inbred pig in the world, Banna minipig inbred (BMI) was potentially suitable for pig-to-human xenotransplantation due to its clear genetic background and minor individual difference. The aim of the research is to compare the hepatic functions between BMI and human. This will provide better evidence for successful xenotransplantation. The liver is a multifunctional organ that is involved in a number of excretory, synthetic, and metabolic functions. It receives, processes, and stores materials absorbed from the digestive tract. Most of the proteins in plasma, including albumin, a- and β-globulins, clotting factors, and transport proteins, are synthesized by the liver. The liver is also the main site of detoxification of exogenous compounds such as drugs and toxins. Many of these specific hepatic functions may be assessed by laboratory procedures to gain insight the integrity of the liver. In this study, we investigated routine hepatic functions, protein electrophoresis, and binding capacity of proteins, and drug metabolism to compare the capacity of liver synthesis, metabolism, and drainage between BMI and humans. The results showed no significant differences in the concentrations of albumin and globulin synthesized in the liver (α1, α2 and β-globulin). Serum enzyme activities in BMI were higherthan those of humans, and levels of total bilirubin (TB) and direct-reacting bilirubin (DB) of BMI were lower than those of humans. The potential of BMI albumin binding to calcium was higher than that of human. The redox activity of serum ceruloplasmin of BMI was significant higher than that of human (P<0.05). The potential of transferrin for transport iron in BMI was higher than that of human. The concentration of plasma haptoglobin (Hpt) of BMI was lower than that of human. The serotypes of Hpt and the concentrations of each serotype of Hpt were different between BMI and human. The Hpt of BMI could play the physiological role of capturing human hemoglobin (Hb) but the capacity of Hpt of BMI binding human Hb was lower than that of human.The difference of thiopurine S-methyltransferase (TPMT) between human and porcine was compared by measuring the activities of TPMT in 10 healthy subjects, 5 BMI and 5 domestic porcine. Erythrocyte TPMT activities were measured by HPLC by assessing the ability of the enzyme to convert 6-mercaptopurine (6-MP) to 6-methylmercaptopurine (6-MMP) using S-adenosyl-methionine as a methyl donor. TPMT activities were expressed as units (U) per ml with the dimensions: 1U = 1.0nmol of 6-MMP produced per hour per ml of packed erythrocytes. The results showed that TPMT activities of human were significantly higher than those of BMIs and domestic porcine (p<0.05). TPMT activities of human were 17.45±3.62 U/ml PRBC and BMIs 8.73±1.55 U/ml PRBC and domestic porcine 7.65±1.35 U/ml PRBC. In this study, we analyzed hepatic coagulant, fibrinolytic, and anticoagulant functions between BMI and human to evaluate such hepatic compatibility. BMI coagulation factors were prepared as the correct testings. BMI factors II,Ⅴ, Ⅶ,Ⅹ, and Ⅻ were added to the corresponding factor-deficient human plasma to determine prothrombin times (PT) and activated partial thromboplastin times (APTT). The results showed that both PT and APTT were reduced but within normal parameters when BMI factors Ⅱ, Ⅴ, Ⅶ, Ⅹ, and Ⅻ were added to the corresponding factor-deficient human plasma. The activities of BMI coagulation factors Ⅱ, Ⅴ, Ⅶ, Ⅸ, and Ⅻ were 3.2, 3.7, 4.7, 2.9, and 4.5 times those of human, respectively. Human tissue plasminogen activator (t-PA) was added to both BMI and human plasma to determine plasmin activity. The activity of plasmin was significantly higher in BMI plasma than in human when human t-PA was added to both. The anti-thrombin-Ⅲ (AT-Ⅲ) activity of plasma was analyzed with the STA-Stago autoanalyzer using an AT-Ⅲ assay kit. The normal range of human AT-Ⅲ activity was 90-108% while BMI AT-Ⅲ was 124.50 ±2.38%. We compared the genetypes of BMI coagulation factor Ⅻ with that of human. The product of RT-PCR of BMI coagulation factor Ⅻ was sequenced. The results showed that the partly gene of BMI coagulation factor Ⅻ showed 85.6%, 81.9%, 79.6% and 78.5% nucleotide identity and 78.9%, 73.3%, 72.2% and 71.1% amino acid identity to human, cavia, rat and mice coagulation factor Ⅻ.Conclusion No significant differences in the concentrations of albumin and globulin synthesized in the liver (α1,α2 and β-globulin) between BMI and humans. These suggested that BMI liver was similar to human in albumin and α, β-globulin synthesis but stronger in bilirubin evacuation, enzyme activity. The capacity of BMI albumin transport calcium was higher than that of human (P<0.05). The redox activity of serum ceruloplasmin of BMI wassignificant higher than that of human (P<0.05). The potential of transferrin for transport iron in BMI was higher than that of human (P<0.05). The Hpt of BMI could play the physiological role of capturing human Hb. And the capacity of BMI Hpt binding human Hb was lower than that of human. TPMT activities of BMI and domestic porcine were significantly lower than those of human. The differences of TPMT between human and porcine may be advisable before institution of thiopurine drugs therapy after porcine livers and kidneys were transplanted into human. BMI liver could have stronger function compared with that of human, and no incompatibility was identified in hepatic function between BMI and human. The activities of coagulation factors and AT-Ⅲ were higher in BMI than in human. BMI coagulation factors Ⅻ, Ⅶ, and Ⅹ trigger human intrinsic, extrinsic and common pathways, respectively, which functioned normally. In addition, BMI plasminogen could be activated by human t-PA.