DMN is a co-inventor on United States Patent Application US-2010-0317597, entitled “Glycomacropeptide (GMP) medical foods for nutritional management of phenylketonuria (PKU) and other metabolic disorders,” 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. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: PS SGM RDB DMN. Performed the experiments: PS SGM SJL RDB DMN. Analyzed the data: PS SGM RDB DMN. Contributed reagents/materials/analysis tools: SJL RDB DMN. Wrote the paper: PS RDB DMN.
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.
Wild type (WT;
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.
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.
Phenylketonuria (PKU; OMIM 261600) is a recessive genetic disease of amino acid (AA) metabolism caused by loss of function mutations of the gene encoding phenylalanine hydroxylase (EC 1.14.16.1,
Compliance with the low-phe diet is often poor after early childhood owing to limited food choices and the bitter taste and strong odor of AA formulas
In order to distinguish the contributions of the PKU genotype itself and dietary treatment of the disease, we have conducted a factorial experiment in PKU (
The University of Wisconsin-Madison Institutional Animal Care and Use Committee approved the facilities and protocols used in this study. A breeding colony of PKU mice was used to produce experimental animals by breeding C57BL/6J mice heterozygous for the
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).
Mice were fed the experimental diets from weaning through young adulthood (3–25 weeks of age), which resulted in mice being fed diet for 20.4±0.11 weeks on average (range 17–22 weeks of feeding, n = 217 mice). The casein, AA and GMP diets were isoenergetic and the source of protein was the only variable manipulated (Harlan Teklad, Madison, WI; TD.09667 - TD.09669), as previously reported
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Variable | Casein | AA | GMP | Casein | AA | GMP | Casein | AA | GMP | Casein | AA | GMP |
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17 (10) | 18 (7) | 19 (7) | 23 (9) | 16 (4) | 22 (10) | 18 (7) | 16 (5) | 14 (6) | 19 (6) | 20 (4) | 13 (3) |
Body Mass (g) |
31.1±0.8 | 28.5±0.5 | 28.1±0.8 | 22.5±0.3 | 22.5±0.3 | 21.9±0.4 | 26.2±0.4 | 29.5±1.0 | 26.8±0.7 | 21.4±0.4 | 22.0±0.3 | 21.5±0.5 |
51.5±4.5 | 38.5±7.0 | 41.0±9.0 | 50.2±1.6 | 34.3±6.1 | 41.9±2.2 | 2026±151 | 726±61 | 745±26 | 2206±76 | 719±19 | 786±24 | |
BMC (mg) |
574±13 | 533±12 | 528±13 | 533±18 | 527±15 | 602±14 | 571±16 | 504±14 | 555±15 | 489±16 | 495±11 | 542±8 |
BMD (mg/cm2) |
51.4±0.3 | 50.7±0.4 | 50.2±0.6 | 51.6±0.6 | 51.4±0.6 | 52.2±0.4 | 49.8±0.3 | 50.1±0.7 | 48.2±0.6 | 48.5±0.5 | 50.4±0.3 | 49.9±0.6 |
Length (mm) |
16.91±0.01 | 16.86±0.02 | 16.91±0.01 | 16.89±0.02 | 16.84±0.02 | 16.88±0.01 | 16.86±0.01 | 16.84±0.03 | 16.85±0.01 | 16.83±0.01 | 16.87±0.02 | 16.81±0.01 |
Values are means ± SE;
Data analyzed by 3 way ANOVA on ranked data.
genotype effect,
sex effect,
diet effect,
gt*sex effect,
gt*diet effect,
sex*diet effect,
gt*sex*diet effect.
Dual-energy x-ray absorptiometry (DXA) with PIXImus software version 2.10 (GE/Lunar Corp, Madison, WI) was performed to obtain in vivo whole body bone mineral density (BMD) and bone mineral content (BMC) at the end of the experiment. Mice were anesthetized with isoflurane with an anesthesia machine (IsoFlo, Abbott Laboratories, North Chicago, IL) and placed prone on the DXA scanner bed with their tail and appendages fully extended. Each mouse received one scan at the completion of the study. Handling of the data obtained from the DXA scan was performed by a single scientist blinded to the treatment groups and subsequent statistical analysis of the densitometry data was performed by the authors. Once mice completed the feeding study, they were placed under anesthesia using an isoflurane anesthesia machine and euthanized by exsanguination via cardiac puncture. Following euthanasia, both femora were dissected free of soft tissue, wrapped in phosphate buffered saline-saturated gauze, and stored at −80°C. Specimens were subjected to two freeze thaw cycles, one prior to DXA, and the second prior to biomechanical testing. Using DXA we measured areal BMD of isolated femura twice with repositioning, as previously described
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.
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Variable | Casein | AA | GMP | Casein | AA | GMP | Casein | AA | GMP | Casein | AA | GMP |
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17 | 18 | 19 | 23 | 16 | 22 | 18 | 16 | 14 | 19 | 20 | 13 |
0.48±0.04 | 0.51±0.07 | 0.55±0.08 | 0.38±0.04 | 0.45±0.04 | 0.40±0.03 | 0.47±0.05 | 0.43±0.06 | 0.46±0.07 | 0.38±0.05 | 0.31±0.03 | 0.30±0.04 | |
0.69±0.04 | 0.74±0.07 | 0.78±0.07 | 0.57±0.04 | 0.65±0.04 | 0.61±0.04 | 0.69±0.05 | 0.65±0.06 | 0.70±0.06 | 0.61±0.05 | 0.51±0.03 | 0.55±0.04 | |
87±4 | 80±2 | 78±4 | 110±5 | 94±5 | 102±3 | 78±4 | 74±4 | 79±7 | 75±2 | 89±3 | 81±3 | |
13.7±0.6 | 13.5±0.3 | 12.9±0.4 | 15.2±0.5 | 14.8±0.4 | 15.2±0.4 | 11.7±0.4 | 12.3±0.4 | 12.6±0.4 | 13.0±0.5 | 13.2±0.4 | 14.3±0.5 | |
15.8±0.5 | 15.0±0.3 | 14.6±0.4 | 18.1±0.5 | 16.6±0.4 | 17.6±0.4 | 14.1±0.4 | 14.1±0.4 | 14.6±0.6 | 14.8±0.4 | 15.3±0.3 | 16.2±0.4 | |
7.54±0.37 | 7.01±0.55 | 7.15±0.61 | 6.87±0.44 | 7.09±0.39 | 7.33±0.46 | 6.61±0.50 | 5.86±0.50 | 6.56±0.65 | 5.90±0.53 | 5.31±0.30 | 5.71±0.37 |
Values are means ± SE of raw data;
Data analyzed using ANCOVA with a covariate of body mass.
Data transformed to satisfy assumptions of normality and variance.
Non-transformable data was ranked.
genotype effect,
sex effect,
diet effect,
gt*sex effect,
gt*sex*diet effect.
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Variable | Casein | AA | GMP | Casein | AA | GMP | Casein | AA | GMP | Casein | AA | GMP |
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17 | 18 | 19 | 23 | 16 | 22 | 18 | 16 | 14 | 19 | 20 | 13 |
1.07±0.04 | 0.97±0.03 | 0.97±0.02 | 1.01±0.02 | 0.98±0.04 | 0.96±0.02 | 0.96±0.02 | 0.95±0.03 | 1.00±0.04 | 0.90±0.02 | 0.91±0.02 | 0.94±0.02 | |
6.07±0.12 | 5.78±0.12 | 5.80±0.10 | 5.60±0.05 | 5.47±0.08 | 5.37±0.04 | 5.80±0.07 | 5.60±0.09 | 5.72±0.12 | 5.49±0.07 | 5.34±0.06 | 5.38±0.07 | |
0.93±0.03 | 0.90±0.02 | 0.91±0.02 | 0.89±0.01 | 0.85±0.01 | 0.84±0.01 | 0.92±0.01 | 0.88±0.02 | 0.87±0.01 | 0.93±0.01 | 0.86±0.01 | 0.88±0.02 | |
1.68±0.05 | 1.60±0.05 | 1.64±0.04 | 1.48±0.02 | 1.46±0.02 | 1.41±0.02 | 1.62±0.03 | 1.54±0.03 | 1.49±0.04 | 1.49±0.03 | 1.41±0.02 | 1.41±0.03 | |
1.34±0.03 | 1.27±0.02 | 1.29±0.02 | 1.32±0.01 | 1.27±0.02 | 1.27±0.01 | 1.29±0.01 | 1.25±0.02 | 1.27±0.02 | 1.31±0.01 | 1.24±0.01 | 1.28±0.02 | |
2.25±0.05 | 2.11±0.05 | 2.11±0.03 | 2.02±0.02 | 1.97±0.03 | 1.93±0.02 | 2.11±0.03 | 2.06±0.04 | 2.10±0.05 | 1.95±0.03 | 1.90±0.03 | 1.92±0.02 | |
1.68±0.02 | 1.66±0.03 | 1.64±0.02 | 1.54±0.02 | 1.55±0.02 | 1.52±0.01 | 1.64±0.02 | 1.65±0.03 | 1.65±0.02 | 1.49±0.01 | 1.53±0.02 | 1.50±0.02 | |
0.19±0.01 | 0.16±0.01 | 0.17±0.01 | 0.17±0.01 | 0.16±0.01 | 0.15±0.00 | 0.16±0.01 | 0.15±0.01 | 0.16±0.01 | 0.16±0.01 | 0.14±0.00 | 0.15±0.01 | |
54.0±0.7 | 51.9±0.6 | 51.8±0.7 | 55.0±0.8 | 54.1±0.9 | 55.0±0.5 | 50.8±0.6 | 50.5±0.9 | 51.1±0.8 | 49.8±0.7 | 51.2±0.4 | 51.3±0.7 | |
BMC (mg) |
29.2±0.6 | 27.3±0.5 | 27.0±0.6 | 27.8±0.6 | 27.1±0.5 | 27.5±0.4 | 26.0±0.5 | 25.5±0.8 | 25.6±0.7 | 23.3±0.6 | 24.6±0.3 | 24.4±0.7 |
Values are means ± SE of raw data;
Data analyzed using ANCOVA with a covariate of body mass.
Data transformed to satisfy assumptions of normality and variance.
Non-transformable data was ranked.
genotype effect,
sex effect,
diet effect,
gt*sex effect,
gt*diet effect.
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Variable | Casein | AA | GMP | Casein | AA | GMP | Casein | AA | GMP | Casein | AA | GMP |
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17 | 18 | 19 | 23 | 16 | 22 | 18 | 16 | 14 | 19 | 20 | 13 |
0.030±0.002 | 0.031±0.002 | 0.031±0.002 | 0.028±0.001 | 0.028±0.001 | 0.028±0.001 | 0.031±0.002 | 0.029±0.002 | 0.032±0.002 | 0.031±0.001 | 0.026±0.001 | 0.034±0.003 | |
0.068±0.005 | 0.070±0.011 | 0.076±0.012 | 0.053±0.005 | 0.061±0.005 | 0.054±0.005 | 0.064±0.007 | 0.058±0.008 | 0.061±0.009 | 0.054±0.008 | 0.041±0.004 | 0.041±0.006 | |
0.098±0.006 | 0.101±0.011 | 0.107±0.011 | 0.080±0.006 | 0.089±0.006 | 0.083±0.005 | 0.095±0.007 | 0.087±0.008 | 0.093±0.008 | 0.085±0.008 | 0.067±0.003 | 0.075±0.005 | |
4184±256 | 4543±234 | 4305±231 | 5670±202 | 5374±223 | 6017±184 | 4236±253 | 4386±173 | 4520±274 | 4314±145 | 5768±234 | 4915±281 | |
92±3 | 102±4 | 96±3 | 111±3 | 115±4 | 121±3 | 86±2 | 98±2 | 98±3 | 103±3 | 112±4 | 117±4 | |
105±3 | 113±4 | 109±3 | 131±3 | 129±4 | 141±3 | 104±2 | 112±2 | 113±3 | 118±3 | 130±3 | 133±4 | |
7.14±0.29 | 6.97±0.40 | 7.19±0.51 | 6.95±0.43 | 7.51±0.47 | 7.87±0.45 | 6.74±0.50 | 6.13±0.45 | 6.84±0.63 | 6.39±0.49 | 5.95±0.31 | 6.39±0.45 | |
80±4 | 76±7 | 72±6 | 99±5 | 91±5 | 105±3 | 76±5 | 84±5 | 82±4 | 92±5 | 104±4 | 106±5 |
Values are means ± SE of raw data;
Data analyzed using ANCOVA with a covariate of body mass.
Data transformed to satisfy assumptions of normality and variance.
Non-transformable data was ranked.
genotype effect,
sex effect,
diet effect,
gt*sex effect,
gt*diet effect,
sex*diet effect,
gt*sex*diet effect.
We tested femoral diaphysis biomechanical performance by quasi-static 3-point bending under displacement control at a rate of 0.3 mm/sec, with a support span of 7.5 mm as previously described,
Stress (σ), (MPa) = FLc/4
Strain (ε), (mm/mm) = 12cd/L2 with c = outer radius in the plane of bending, d = displacement, L = length.
Young’s Modulus (E), (MPa) = (F/d)(L3/48
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).
The following input variables were included in principal component analysis (PCA): Body mass, femoral BMD, post-yield deflection, total deflection, yield load, maximum load, energy, stiffness, femoral cross-sectional area, femoral periosteal perimeter, femoral inner major and minor axis lengths, femoral outer major and minor axis lengths, femoral diaphyseal shape factor, and femoral diaphyseal cross-sectional moment of inertia. The PCA was performed with the SAS function “proc princomp” (SAS Institute, Cary, NC)
Mass | CSA | Perim | InMin | InMaj | OutMin | OutMaj | SF | CSMI | PYD | TD | Stiff | YLoad | MLoad | Energy | fBMD | |
Mass | – | 0.45 | 0.63 | 0.30 | 0.56 | 0.29 | 0.67 | 0.61 | 0.47 | 0.27 | 0.29 | −0.15 | −0.08 | −0.10 | 0.22 | 0.14 |
CSA | – | 0.73 | 0.23 | 0.33 | 0.65 | 0.71 | 0.36 | 0.85 | 0.23 | 0.24 | 0.39 | 0.44 | 0.51 | 0.40 | 0.64 | |
Perim | – | 0.67 | 0.80 | 0.74 | 0.97 | 0.61 | 0.87 | 0.30 | 0.33 | −0.01 | 0.11 | 0.10 | 0.29 | 0.31 | ||
InMin | – | 0.70 | 0.78 | 0.58 | 0.09 | 0.65 | 0.10 | 0.10 | −0.07 | −0.01 | 0.01 | 0.03 | 0.06 | |||
InMaj | – | 0.53 | 0.82 | 0.60 | 0.64 | 0.34 | 0.36 | −0.16 | −0.08 | −0.12 | 0.22 | 0.11 | ||||
OutMin | – | 0.63 | −0.04 | 0.91 | 0.13 | 0.14 | 0.25 | 0.33 | 0.40 | 0.22 | 0.42 | |||||
OutMaj | – | 0.75 | 0.79 | 0.31 | 0.35 | −0.02 | 0.08 | 0.06 | 0.30 | 0.31 | ||||||
SF | – | 0.25 | 0.29 | 0.33 | −0.22 | −0.17 | −0.25 | 0.19 | 0.05 | |||||||
CSMI | – | 0.26 | 0.27 | 0.28 | 0.35 | 0.42 | 0.36 | 0.53 | ||||||||
PYD | – | 0.98 | 0.00 | −0.05 | −0.02 | 0.86 | 0.11 | |||||||||
TD | – | −0.10 | −0.02 | −0.05 | 0.84 | 0.09 | ||||||||||
Stiff | – | 0.46 | 0.73 | 0.20 | 0.68 | |||||||||||
YLoad | – | 0.85 | 0.30 | 0.67 | ||||||||||||
MLoad | – | 0.36 | 0.76 | |||||||||||||
Energy | – | 0.39 | ||||||||||||||
fBMD | – |
Phenotypes are Mass, body mass; CSA, cortical cross-sectional area; Perim, periosteal perimeter; InMin, inner minor axis; InMaj, inner major axis; OutMin, outer minor axis; OutMaj, outer major axis; SF, shape factor; CSMI, cross-sectional moment of inertia; PYD, post-yield deflection; TD, total deflection; Stiff, stiffness; YLoad, yield load; MLoad, maximum load; Energy, energy to failure; fBMD, BMD by ex vivo DXA. Each cell shows R.
PC1 | PC2 | PC3 | PC4 | |
Eigenvalue | 6.68 | 3.57 | 2.39 | 1.29 |
Difference | 3.11 | 1.19 | 1.09 | 0.72 |
r2 | 0.418 | 0.223 | 0.149 | 0.081 |
Cumulative r2 | 0.418 | 0.641 | 0.79 | 0.871 |
Difference is subtraction of subsequent PC from former PC.
Data were analyzed by three-way ANOVA or ANCOVA using “proc mixed”. Femoral biomechanical testing was performed on two different days due to the large number of femora, thus a random effect of time as a blocking factor was included in the model. The three-way ANCOVA tested for main effects of genotype, sex, and diet as well as their two and three way interactions. Femoral cross section measurements and biomechanical data were adjusted for the animal’s body mass by including body mass as a covariate. When body mass wasn’t a significant predictor for a parameter the term was removed and results from a subsequent three-way ANOVA are presented. Data presented are raw data or actual measurements, and the statistical significance shown represents the analysis adjusted for body weight based on ANCOVA where appropriate. Differences between treatment groups were detected using a protected Fisher's Least Significant Difference (LSD) test (SAS Institute, 2007, Cary, NC). Data transformations were performed where appropriate to fit assumptions of normality and equal variance prior to statistical analysis. If data transformations failed, a respective non-parametric ANCOVA or ANOVA was performed on ranked data. Untransformed data are presented in the tables. Data are analyzed per animal; biomechanical data are an average of the right and left femora. Data are presented as mean ± SE. P-values <0.05 are considered significant. Where there was no significant interaction, data were pooled into treatment groups by their respective significant main effects.
PC1 | PC2 | PC3 | PC4 | |
Body mass | 0.239 | −0.205 | −0.026 | 0.318 |
Cross-sectional area | 0.316 | 0.161 | −0.025 | 0.227 |
Perimeter | 0.358 | −0.125 | −0.147 | 0.040 |
Inner minor axis | 0.234 | −0.111 | −0.296 | −0.474 |
Inner major axis | 0.289 | −0.242 | −0.116 | −0.077 |
Outer minor axis | 0.302 | 0.107 | −0.230 | −0.375 |
Outer major axis | 0.350 | −0.153 | −0.112 | 0.171 |
Shape factor | 0.194 | −0.282 | 0.053 | 0.538 |
CSMI | 0.358 | 0.081 | −0.143 | −0.122 |
Post-yield deflection | 0.183 | −0.125 | 0.518 | −0.203 |
Total deflection | 0.189 | −0.147 | 0.507 | −0.186 |
Stiffness | 0.083 | 0.409 | 0.036 | 0.074 |
Yield load | 0.123 | 0.407 | 0.028 | 0.071 |
Max load | 0.136 | 0.468 | 0.035 | 0.027 |
Energy | 0.209 | 0.063 | 0.506 | −0.116 |
Femoral BMD | 0.209 | 0.362 | 0.051 | 0.202 |
CSMI, cross-sectional moment of inertia; BMD, bone mineral density.
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Variable | Casein | AA | GMP | Casein | AA | GMP | Casein | AA | GMP | Casein | AA | GMP |
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17 | 18 | 19 | 23 | 16 | 22 | 18 | 16 | 14 | 19 | 20 | 13 |
25.1±0.6 | 23.4±0.4 | 23.1±0.6 | 24.8±0.6 | 23.2±0.5 | 23.9±0.4 | 22.2±0.5 | 22.4±0.6 | 22.5±0.8 | 20.7±0.4 | 21.8±0.3 | 21.4±0.4 | |
40.8±1.7 | 38.2±1.1 | 37.0±2.0 | 53.7±2.4 | 46.3±2.1 | 50.5±1.5 | 36.4±1.9 | 34.3±1.7 | 37.4±3.1 | 37.2±1.1 | 43.1±1.1 | 40.9±1.4 | |
5.75±0.23 | 5.41±0.36 | 5.42±0.47 | 6.58±0.35 | 6.20±0.20 | 6.65±0.30 | 5.04±0.36 | 4.47±0.38 | 5.12±0.53 | 4.75±0.34 | 4.94±0.23 | 4.93±0.25 | |
17.3±0.4 | 15.9±0.2 | 15.6±0.4 | 16.4±0.4 | 15.1±0.4 | 15.5±0.3 | 14.9±0.4 | 15.8±0.5 | 15.3±0.6 | 13.3±0.3 | 14.7±0.2 | 14.0±0.4 |
Values are means ± SE;
Data transformed to satisfy assumptions of normality and variance.
genotype effect,
sex effect,
gt*sex,
gt*diet effect,
gt*sex*diet effect.
The low-phe AA and GMP diets significantly reduced plasma phe concentration in both WT and PKU mice,
The 3-point bending test produces mid-diaphyseal fractures under controlled loading conditions (
Regardless of sex or diet, the PKU genotype was associated with reduced femoral biomechanical properties assessed by three-point bending
We sought evidence regarding whether plasma phe levels are related to bone status. We found no significant relationship between plasma phe and any biomechanical or BMD outcomes (data not shown).
Regardless of genotype and sex, the AA diet reduced femoral size, as manifested in a significant reduction in femoral cross sectional area (CSA) and in the femoral perimeter, compared with the casein and GMP diets (
The bone properties we measured in this study are not mutually independent. For example, yield load and maximum load are highly correlated with an R of 0.85 (
The PCs are composites of all the measured parameters, but can be interpreted on the basis of the coefficients for each, as summarized in the Eigenvectors (
Success in managing the neurological manifestations of PKU by dietary phe restriction has allowed many patients to enjoy a greatly improved prognosis, spared of devastating cognitive impairment. Because of their improved function, other, more subtle deficits have become apparent
Our data show that PKU mice have impaired bone biomechanical performance, regardless of the effects of sex or diet. The biomechanical deficit is complex, encompassing strength, stiffness, and ductility. PCA reveals a genotype effect for all 4 PCs, confirming the principal findings of the whole bone biomechanics. Bone is a composite tissue composed of a protein matrix, containing primarily type 1 collagen, and precipitated mineral, containing primarily calcium and phosphate in the form of apatite
While our data clearly support the existence of deficits in bone strength and ductility in PKU mice, the present data are insufficient to identify the biochemical, cellular, and physiological mechanisms underlying them. The organic components of bone matrix are produced by osteoblasts and these are mineralized following secretion into the extracellular space
It is important to recall that bone is known to model in response to its usual loading environment, and bone size and strength therefore vary as a function of body size and activity
Our data also demonstrate that dietary protein source consistently affects both size and strength in WT and PKU mice. The AA diet impairs the radial growth of the femur, affecting all diaphyseal dimensions relative to casein (
The work reported here can only be compared to prior studies in limited ways. A prior experiment reported that 8 weeks of phe restriction improved bone status in PKU mice
Our experiment featured several notable strengths. Use of a uniform C57BL/6J genetic background eliminated possible confounding due to the segregation of genes other than
Several limitations of our work must also be acknowledged. The C57BL/6 background is inbred, and therefore not reflective of the varied, outbred genetic backgrounds encountered in human PKU. The 3 point bending test, while robust and reproducible, produces experimental fractures in the mid-diaphysis of the femur, a site that is not generally susceptible to clinical fracture. Moreover, this is a skeletal site that is composed of cortical bone, so our data do not address the consequences of PKU on trabecular bone. Mice, because of their small size, do not have the osteonal structure characteristic of human cortical bone. The diets were only started at 3 weeks of age, when mice are weaned. This is distinct from PKU management in humans, which features dietary restriction beginning in the first week following birth. However, since long bones in mice undergo extensive linear and radial growth between weaning and young adulthood, most, if not all the femoral diaphyseal bone present at the time of testing was synthesized during the course of study. Finally, while our study was adequately powered to detect genotype, diet, and sex differences in all aspects of biomechanical performance at the main effect level, the sample sizes were insufficient to detect the impact of interactions on energy, post-yield displacement, and their material level correlates.
In summary, the data reported here demonstrate that skeletal fragility is an intrinsic feature of PKU in mice. The biomechanical defects are complex, affecting both strength and ductility. In mice, an AA diet exacerbates skeletal fragility by limiting radial bone growth, which is attenuated by a GMP diet. As an AA diet is presently the standard of care, this finding suggests that there is a need to determine whether improved diets can improve bone health in patients with PKU. The mechanisms by which PKU causes bone fragility and the AA-based diet impairs radial bone growth remain unknown, illustrating the need for further work to fully define the skeletal pathophysiology of PKU and its treatment.
We thank Dr. Adam Brinkman for his assistance with tissue processing and review of the manuscript, graduate student Emily Sawin for assistance with data analysis, undergraduate students Wing Pun and Jennifer Mallon for their assistance with the PKU mouse colony, and Peter Crump for assistance with statistical models.