Low bone strength is a manifestation of phenylketonuria in mice and is attenuated by a glycomacropeptide diet

Abstract

Purpose: Phenylketonuria (PKU), caused by phenylalanine (phe) hydroxylase loss of function mutations, requires a low-phe diet plus amino acid (AA) formula to prevent cognitive impairment. Glycomacropeptide (GMP), a low-phe whey protein, provides a palatable alternative to AA formula. Skeletal fragility is a poorly understood chronic complication of PKU. We sought to characterize the impact of the PKU genotype and dietary protein source on bone biomechanics.

Procedures: Wild type (WT; Pah(+/+)) and PKU (Pah(enu2/enu2)) mice on a C57BL/6J background were fed high-phe casein, low-phe AA, and low-phe GMP diets between 3 to 23 weeks of age. Following euthanasia, femur biomechanics were assessed by 3-point bending and femoral diaphyseal structure was determined. Femoral ex vivo bone mineral density (BMD) was assessed by dual-energy x-ray absorptiometry. Whole bone parameters were used in principal component analysis. Data were analyzed by 3-way ANCOVA with genotype, sex, and diet as the main factors.

Findings: Regardless of diet and sex, PKU femora were more brittle, as manifested by lower post-yield displacement, weaker, as manifested by lower energy and yield and maximal loads, and showed reduced BMD compared with WT femora. Four principal components accounted for 87% of the variance and all differed significantly by genotype. Regardless of genotype and sex, the AA diet reduced femoral cross-sectional area and consequent maximal load compared with the GMP diet.

Conclusions: Skeletal fragility, as reflected in brittle and weak femora, is an inherent feature of PKU. This PKU bone phenotype is attenuated by a GMP diet compared with an AA diet.

Figure 1. Experimental design and three-point bending test.

The experiment utilized a 2×2×3 factorial design with a total of 12 groups (A). A cartoon of the three-point bending test of a mouse femur and a representative photograph (B).

Figure 2. Force-displacement curve analysis of WT and PKU mice.

Schematic of a load-displacement curve generated from the three-point bending test from which the yield point, maximum load, elastic and plastic deformation, and energy to failure (shaded area under the curve) are obtained (A). Representative load-displacement curves for WT and PKU mice (B). Effects in WT and PKU mice for yield load (C), maximum load (D), post-yield displacement (PYD) (E), total displacement (F), energy to failure (G), and femoral bone mineral density (BMD) (H). Values are means ± SE; p-values represent main effect of genotype. Sample size is shown in parenthesis. All values for femoral biomechanical performance had a significant main effect for genotype, WT >PKU.

Figure 3. Diet modifies femoral size and strength in WT and PKU mice.

Representative photographs of femoral cross-sectional geometry in mice fed casein, AA, and GMP diets from which measurements of cross-sectional area and perimeter are obtained (A). Diet effect on maximum load derived from load-displacement curve analysis (B). Values are means ± SE; p-values represent main effect of diet. Sample size is shown in parenthesis. Groups with different letter superscripts are significantly different (p<0.05).