Low therapeutic threshold for hepatocyte replacement in murine phenylketonuria

Abstract

Phenylalanine homeostasis in mammals is primarily controlled by liver phenylalanine hydroxylase (PAH) activity. Inherited PAH deficiency (phenylketonuria or PKU) leads to hyperphenylalaninemia in both mice and humans. A low level of residual liver PAH activity ensures near-normal dietary protein tolerance with normal serum phenylalanine level, but the precise threshold for normal phenylalanine clearance is unknown. We employed hepatocyte transplantation under selective growth conditions to investigate the minimal number of PAH-expressing hepatocytes necessary to prevent hyperphenylalaninemia in mice. Serum phenylalanine levels remained normal in mice exhibiting nearly complete liver repopulation with PAH-deficient hepatocytes (<5% residual wild-type liver PAH activity). Conversely, transplantation of PAH-positive hepatocytes into PAH-deficient Pah(enu2) mice, a model of human PKU, yielded a significant decrease in serum phenylalanine (<700 muM) when liver repopulation exceeded approximately 5%. These data suggest that restoration of phenylalanine homeostasis requires PAH activity in only a minority of hepatocytes.

Fig. 1

FAH immunohistology of FAH-deficient recipient livers following hepatocyte transplantation. (A and B) Fah^Δexon5/Fah^Δexon5 liver following transplantation with PAH-deficient Pah^enu2/Pah^enu2 hepatocytes. (A) Liver exhibiting extensive repopulation with FAH-positive (but PAH-negative) cells 35 days after transplant. (B) Liver from Mouse 2 (Table 1) demonstrating nearly complete repopulation with FAH-positive cells 117 days after hepatocyte transplant and a second round of regeneration induced by partial hepatectomy. A few FAH-deficient hepatocytes remain in the transplant-recipient liver (arrows). (C and D) Pah/Fah liver following transplantation with wild-type FAH-positive, PAH-positive hepatocytes. (C) Liver from Mouse F (Table 2) containing discrete islands of FAH-positive hepatocytes. The repopulation frequency measured 6.3 ± 1.0% by PAH assay. (D) Liver of Mouse A (Table 2) demonstrating more extensive repopulation (20.5 ± 1.8% by PAH assay) with FAH-positive wild-type hepatocytes.

Fig. 2

Plot of liver PAH activity in transplanted Fah^Δexon5/Fah^Δexon5 mice vs time in days after transplant. The extent of repopulation increased and hence PAH activity (nmol Tyr produced/h/mg protein) decreased with time in animals that received PAH-deficient Pah^enu2/Pah^enu2 hepatocytes.

Fig. 3

Serum phenylalanine vs time in Pah/Fah mice after wild-type hepatocyte transplant. Serum phenylalanine levels in Pah/Fah mice are plotted vs days after transplant with wild-type hepatocytes. Serum phenylalanine levels corrected as early as 22 days and as late as 69 days after transplant. Normal range of serum phenylalanine is depicted in the gray-shaded area.

Fig. 4

Comparison of serum phenylalanine to extent of liver repopulation in transplanted Pah/Fah mice. Serum Phe levels for transplanted mice at the time of tissue harvest are plotted against % wild-type PAH activity measured in liver homogenate or isolated hepatocytes. Reasoning that Phe clearance would have been completely corrected in the single animal with >10% wild-type PAH activity, regression analysis examining the relationship between serum Phe and liver PAH activity was performed using only mice with <10% wild-type PAH activity (Mice B–K).