1. Effects of Glycomacropeptide, Amino Acid & Casein Diets on Osteopenia in PKU Mice Denise M. Ney, PhD, RD Professor of Nutritional Sciences Waisman Center University of Wisconsin-Madison

2. Disclosure D Ney is a co-inventor on US Patent Application US-2010-0317597, GMP Medical Foods for Nutritional Management of PKU, which is held by the Wisconsin Alumni Research Foundation and licensed to Cambrooke Foods, LLC. A percentage of all royalty payments is awarded to the inventors. D Ney has received consulting income from Cambrooke Foods and BioMarin.

3. Funding: National PKU Alliance & USDA Hatch Grant Denise Ney Robert Blank Sangita Murali Patrick Solverson UW-Madison PKU Bone Research Team

4. Background PKU is associated with low bone mass, or osteopenia, and fractures in early adulthood. –57% of 28 patients had osteopenia/osteoporosis Perez-Dueñas et al. Acta Paediatr 91:800, 2002 Reduced bone mineral density (BMD) in PKU is present from an early age onward and it cannot be predicted by plasma phe levels. –20% of 53 patients studied had osteoporosis de Groot et al. Mol Genet Metab 101:566, 2012

5. What causes skeletal fragility in PKU? The fundamental question is whether reduced BMD is inherent to PKU or secondary to its dietary management. In order to isolate the contributions of the PKU genotype itself and dietary treatment of PKU we have conducted a factorial experiment in PKU mice.

6. Objective & Design To determine how the PKU genotype and the source of dietary protein affect growth, body composition and bone development. WT PKU AA High Phe Low Phe Casein GMP

7. PKU & WT Mice Housed With Same Sex Littermates phe GMP phe AA phe Casein

8. GMP is a natural whey protein produced when making cheese. Pure GMP contains no phe.

9. Growth Solverson, P et al Am J Physiol Endocrinol Metab 302:E885-95, 2012

10. Metabolic Phenotyping Platform Food & water intake O 2 consumption & CO 2 production

11. PKU mice show increased energy expenditure with casein diet

12. Values with different letter superscripts are significantly different, p<0.05 GMP Diet Normalizes Food Intake in PKU Mice

13. AA Diet Increases Kidney Workload in both WT and PKU Mice Values with different letter superscripts are significantly different, p<0.05

14. Does PKU increase energy needs? Resting energy expenditure is ~5-10% higher in adolescent females with PKU than that predicted by standard equations. –J Am Diet Assoc 110:922-25, 20101 –Am J Clin Nutr 62:797-804, 1995 Reduced growth occurs in children with PKU –Mol Genet Metab 101:99-109, 2010 –J Pediatr 26:1-11, 2002

15. Dual Energy X-Ray Absorptiometry (DXA)

16. PKU mice show reduced whole-body bone mineralization compared to WT mice

17. Femur Strength: 3 Point Loading Test Loading force being applied to a mouse femur Displacement Load (F)

18. Displacement, mm Load-Displacement Curve 0.3 -2-2 -0.1 0.10.20.40.50.60.70.8 Load, N Fracture Yield Point Elastic Deformation Post-Yield Displacement 12 10 8 6 4 2 14 16 Max Load

19. PKU mice show bones that are brittle and break easily

20. PKU mice show reduced femoral bone mineralization compared to WT mice

21. How the casein, AA and GMP diets affect bone size and strength?

22. GMP Increases Bone Size in PKU & WT Mice AA Casein GMP

23. GMP Improves Bone Strength in PKU and WT Mice Compared with AA Diet

24. Summary PKU mice have femora that are brittle and weak. This suggests defects in both collagen synthesis and mineralization. GMP increases bone size and strength; femora tolerate a higher max load before fracture compared with the AA diet. –How does GMP work to improve bone strength in mice?

25. What causes skeletal fragility in PKU?

26. Conclusion Skeletal fragility is inherent to the PKU genotype and is attenuated by a GMP compared with an AA diet in mice. Future research is needed to determine if improved low-phe diets containing GMP reduce skeletal fragility in human PKU.

27. Take Home Message: How can bone health be improved in PKU? Follow a low-phe diet to improve growth and bone development Include weight bearing exercise each day