Effects of Glycomacropeptide, Amino Acid & Casein Diets on

Osteopenia in PKU Mice

Denise M. Ney, PhD, RD Professor of Nutritional Sciences

Waisman CenterUniversity of Wisconsin-Madison

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.

Funding: National PKU Alliance & USDA Hatch Grant

Denise Ney Robert Blank Sangita Murali Patrick Solverson

UW-Madison PKU Bone Research Team

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

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.

Objective & Design To determine how the PKU genotype and

the source of dietary protein affect growth, body composition and bone development.

WTPKU

♂♀ AA

High Phe

Low Phe

Casein

GMP

PKU & WT Mice Housed With

Same Sex Littermates

↓phe GMP ↓phe AA ↑phe Casein

GMP is a natural whey protein produced when

making cheese. Pure GMP contains no phe.

Growth

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

Males

3 6 9 12 15 18 21 24

Bo

dy

Wei

gh

t (g

)

5

10

15

20

25

30

35

Age (Week)

Genotype x Diet p = 0.0053

PKU AA (12)b PKU CAS (16)b

PKU GMP (11)b WT AA (13)b WT CAS (15)a

WT GMP (17)b

Females

3 6 9 12 15 18 21 24

Bo

dy

Wei

gh

t (g

)

5

10

15

20

25

Age (Week)

Genotype x Diet p = 0.0257

PKU AA (18)bc PKU CAS (14)c

PKU GMP (11)bc WT AA (14)ab WT CAS (19)a

WT GMP (20)b

Metabolic Phenotyping Platform

●Food & water intake●O2 consumption & CO2 production

PKU mice show increased energy expenditure with

casein diet

Lean Body Mass (g)

12 14 16 18 20 22 24 26

VO

2 (m

l/hr)

60

70

80

90

100

110

120

PKUab

b

a

ccc

WT

3-15%

WTAA (23)

WTCAS (16)

WTGMP (26)

PKUAA (18)

PKUCAS (21)

PKUGMP (16)

0

1

2

3

4

5F

oo

d In

take

(g

/day

)

Wild Type PKU

Cas

ein

(17

)

AA

(23

)

GM

P (

30)

Cas

ein

(21

)

AA

(18

)

GM

P (

17)

genotype x diet p = 0.0193

sex p = 0.0404males - afemales - b a

a

bbbb

Values with different letter superscripts are significantly different, p<0.05

GMP Diet Normalizes Food Intake in PKU

Mice

AA Diet Increases Kidney Workload in both WT and PKU

Mice

Values with different letter superscripts are significantly different, p<0.05

24 Hour Water Intake

24 h

r W

ater

Inta

ke (

ml)

0

2

4

6

8

WT PKU

Cas

ein

(20

)

AA

(23

)

GM

P (

30)

Cas

ein

(21

)

AA

(18

)

GM

P (

16)

a a

a

bb b

Kidney Mass

0.0

0.4

0.8

1.2

1.6

2.0

g/1

00g

BW

Wild Type PKU

AA

(27

)

Cas

ein

(34

)

GM

P (

36)

AA

(30

)

Cas

ein

(30

)

GM

P (

22)

c

a

cb ab

c

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

Dual Energy X-Ray Absorptiometry (DXA)

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

miceB

MD

(m

g/c

m2 )

0

10

20

30

40

50

60

WT (98) PKU (86)

a b

Femur Strength: 3 Point Loading Test

Loading force being applied to a mouse

femur

Displacement

Load (F)

Displacement, mm

Load-Displacement Curve

0.3

-2-0.1 0.1 0.2 0.4 0.5 0.6 0.7 0.8

Loa

d, N Fracture

Yield

Point

Elastic Deformation

Post-Yield Displacement

12

10

8

6

4

2

14

16

Max Load

0

2

4

6

8

10

12

14

16

WT (116)

Yie

ld L

oa

d (

N)

PKU (101)

ab

WT (116)

PY

D (

mm

)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

PKU (101)

ab

PKU mice show bones that are brittle and break easily

0.0 0.2 0.4 0.6 0.8

0

2

4

6

8

10

12

14

16

18

20Wild TypePKU

Displacement (mm)

Lo

ad (

N)

Yield Load

Fracture

Post-Yield Displacement

PKU mice show reduced femoral bone mineralization compared to WT mice

0

5

10

15

20

25

30

Bone Mineral Content

WT (116)

BM

C (

mg

)

PKU (101)

ab

0

10

20

30

40

50

60

Bone Mineral Density

WT (116)

BM

D (

mg

/cm

2 )

PKU (101)

a b

How the casein, AA and GMP diets affect bone size

and strength?

GMP Increases Bone Size in PKU & WT Mice

Cross Sectional Area

Casein

(m

m2 )

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

AA GMP

a ab WT MiceFed Casein

AA

Casein

GMP

0

2

4

6

8

10

12

14

16

18

Max

Lo

ad (

N)

Casein AA GMP

aab b WT MiceFed Casein

GMP Improves Bone Strength in PKU and WT Mice Compared with

AA Diet

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?

What causes skeletal fragility in PKU?

Inherent to PKU genotype and/or Secondary to management with an AA

diet.

Answer = both genotype and diet

ConclusionSkeletal 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.

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