Sex effects of dietary protein source and acid load on renal and bone status in the Pahenu2 mouse model of phenylketonuria.

The low-phenylalanine (Phe) diet with amino acid (AA) medical foods is associated with low bone mineral density (BMD) and renal dysfunction in human phenylketonuria (PKU). Our objective was to determine if diets differing in dietary protein source and acid load alter bone and renal outcomes in Pah-/- and wild-type (WT) mice. Female and male Pah-/- (Pahenu2/enu2 ) and WT littermates (C57BL/6 background) were fed high-acid AA, buffered AA (BAA), glycomacropeptide (GMP), or high-Phe casein diets from 3 to 24 weeks of age. The BAA diet significantly reduced the excretion of renal net acid and ammonium compared with the AA diet. Interestingly, the BAA diet did not improve renal dilation in hematoxylin and eosin (H&E) stained renal sections, femoral biomechanical parameters, or femoral bone mineral content (BMC). Significantly lower femoral BMC and strength occurred in Pah-/- versus WT mice, with greater decline in female Pah-/- mice. Polyuria and mild vacuolation in the proximal convoluted tubules were observed in male Pah-/- and WT mice fed the high-acid AA diet versus absent/minimal cortical vacuolation in males fed the GMP, BAA, or casein diets. Vacuole contents in male mice were proteinaceous. Cortical vacuolation was absent in female mice. Dilated medullary tubules were observed in all Pah-/- mice, except for male Pah-/- mice fed the GMP diet. In summary, the PKU genotype and diet showed differential effects on renal and bone status in male and female mice. Renal status improved in male Pah-/- mice fed the GMP diet.

Sex-specific deficits in neurite density and white matter integrity are associated with targeted disruption of exon 2 of the Disc1 gene in the rat.

Diffusion tensor imaging (DTI) has provided remarkable insight into our understanding of white matter microstructure and brain connectivity across a broad spectrum of psychiatric disease. While DTI and other diffusion weighted magnetic resonance imaging (MRI) methods have clarified the axonal contribution to the disconnectivity seen in numerous psychiatric diseases, absent from these studies are quantitative indices of neurite density and orientation that are especially important features in regions of high synaptic density that would capture the synaptic contribution to the psychiatric disease state. Here we report the application of neurite orientation dispersion and density imaging (NODDI), an emerging microstructure imaging technique, to a novel Disc1 svΔ2 rat model of psychiatric illness and demonstrate the complementary and more specific indices of tissue microstructure found in NODDI than those reported by DTI. Our results demonstrate global and sex-specific changes in white matter microstructural integrity and deficits in neurite density as a consequence of the Disc1 svΔ2 genetic variation and highlight the application of NODDI and quantitative measures of neurite density and neurite dispersion in psychiatric disease.

Refractory diet-dependent changes in neural microstructure: Implications for microstructural endophenotypes of neurologic and psychiatric disease.

Alterations in gut microbiome populations via dietary manipulation have been shown to induce diet-dependent changes in white matter microstructure. The purpose of this study is to examine the durability of these diet-induced microstructural alterations. We implemented a crossover experimental design where post-weaned male rats were assigned to one of four experimental diets. Following the administration of experimental diets and again following crossover and resumption of a normal diet, brains were imaged ex-vivo with diffusion tensor imaging. Following standard image preprocessing, tract-based spatial statistics and region-of-interest measurements were then calculated for all diffusion tensor indices. Voxel-wise differences in FA were identified in the high fat diet group when compared to animals receiving a control diet. Following crossover, there were new voxel-wise changes in both FA and TR that do not correspond to the regions previously identified. Animals crossed over from the high fiber diet demonstrate widespread and global changes in the diffusion tensor that stand in stark contrast to the minimal changes identified before crossover. While no significant differences between any of the diffusion metrics were identified in the high protein group before crossover, statistically significant decreased RD values were observed following resumption of a normal diet. Diet-induced changes in neural microstructure are durable changes that are unrecoverable following the resumption of a normal diet. We further show that in certain experimental diets, resumption of a normal diet can lead to further marked and unanticipated changes in white matter microstructure.

Gut microbiome populations are associated with structure-specific changes in white matter architecture.

Altered gut microbiome populations are associated with a broad range of neurodevelopmental disorders including autism spectrum disorder and mood disorders. In animal models, modulation of gut microbiome populations via dietary manipulation influences brain function and behavior and has been shown to ameliorate behavioral symptoms. With striking differences in microbiome-driven behavior, we explored whether these behavioral changes are also accompanied by corresponding changes in neural tissue microstructure. Utilizing diffusion tensor imaging, we identified global changes in white matter structural integrity occurring in a diet-dependent manner. Analysis of 16S ribosomal RNA sequencing of gut bacteria also showed changes in bacterial populations as a function of diet. Changes in brain structure were found to be associated with diet-dependent changes in gut microbiome populations using a machine learning classifier for quantitative assessment of the strength of microbiome-brain region associations. These associations allow us to further test our understanding of the gut-brain-microbiota axis by revealing possible links between altered and dysbiotic gut microbiome populations and changes in brain structure, highlighting the potential impact of diet and metagenomic effects in neuroimaging.

Dietary amino acid intakes associated with a low-phenylalanine diet combined with amino acid medical foods and glycomacropeptide medical foods and neuropsychological outcomes in subjects with phenylketonuria.

This article provides original data on median dietary intake of 18 amino acids from amino acid medical foods, glycomacropeptide medical foods, and natural foods based on 3-day food records obtained from subjects with phenylketonuria who consumed low-phenylalanine diets in combination with amino acid medical foods and glycomacropeptide medical foods for 3 weeks each in a crossover design. The sample size of 30 subjects included 20 subjects with classical phenylketonuria and 10 with a milder or variant form of phenylketonuria. Results are presented for the Delis-Kaplan Executive Function System and the Cambridge Neuropsychological Test Automated Battery; the tests were administered at the end of each 3-week dietary treatment with amino acid medical foods and glycomacropeptide medical foods. The data are supplemental to our clinical trial, entitled "Glycomacropetide for nutritional management of phenylketonuria: a randomized, controlled, crossover trial, 2016 (1) and "Metabolomic changes demonstrate reduced bioavailability of tyrosine and altered metabolism of tryptophan via the kynurenine pathway with ingestion of medical foods in phenylketonuria, 2017 (2). This data has been made public and has utility to clinicians and researchers due to the following: 1) This provides the first comprehensive report of typical intakes of 18 amino acids from natural foods, as well as amino acid and glycomacropeptide medical foods in adolescents and adults with phenylketonuria; and 2) This is the first evidence of similar standardized neuropsychological testing data in adolescents and adults with early-treated phenylketonuria who consumed amino acid and glycomacropeptide medical foods.

Amino Acid Medical Foods Provide a High Dietary Acid Load and Increase Urinary Excretion of Renal Net Acid, Calcium, and Magnesium Compared with Glycomacropeptide Medical Foods in Phenylketonuria.

Background. Skeletal fragility is a complication of phenylketonuria (PKU). A diet containing amino acids compared with glycomacropeptide reduces bone size and strength in mice. Objective. We tested the hypothesis that amino acid medical foods (AA-MF) provide a high dietary acid load, subsequently increasing urinary excretion of renal net acid, calcium, and magnesium, compared to glycomacropeptide medical foods (GMP-MF). Design. In a crossover design, 8 participants with PKU (16-35 y) provided food records and 24-hr urine samples after consuming a low-Phe diet in combination with AA-MF and GMP-MF for 1-3 wks. We calculated potential renal acid load (PRAL) of AA-MF and GMP-MF and determined bone mineral density (BMD) measurements using dual X-ray absorptiometry. Results. AA-MF provided 1.5-2.5-fold higher PRAL and resulted in 3-fold greater renal net acid excretion compared to GMP-MF (p = 0.002). Dietary protein, calcium, and magnesium intake were similar. GMP-MF significantly reduced urinary excretion of calcium by 40% (p = 0.012) and magnesium by 30% (p = 0.029). Two participants had low BMD-for-age and trabecular bone scores, indicating microarchitectural degradation. Urinary calcium with AA-MF negatively correlated with L1-L4 BMD. Conclusion. Compared to GMP-MF, AA-MF increase dietary acid load, subsequently increasing urinary calcium and magnesium excretion, and likely contributing to skeletal fragility in PKU. The trial was registered at clinicaltrials.gov as NCT01428258.

Metabolomic changes demonstrate reduced bioavailability of tyrosine and altered metabolism of tryptophan via the kynurenine pathway with ingestion of medical foods in phenylketonuria.

BACKGROUND: Deficiencies of the monoamine neurotransmitters, such as dopamine synthesized from Tyr and serotonin synthesized from Trp, are of concern in PKU. Our objective was to utilize metabolomics analysis to assess monoamine metabolites in subjects with PKU consuming amino acid medical foods (AA-MF) and glycomacropeptide medical foods (GMP-MF). METHODS: Subjects with PKU consumed a low-Phe diet combined with AA-MF or GMP-MF for 3weeks each in a randomized, controlled, crossover study. Metabolomic analysis was conducted by Metabolon, Inc. on plasma (n=18) and urine (n=9) samples. Catecholamines and 6-sulfatoxymelatonin were measured in 24-h urine samples. RESULTS: Intake of Tyr and Trp was ~50% higher with AA-MF, and AA-MF were consumed in larger quantities, less frequently during the day compared with GMP-MF. Performance on neuropsychological tests and concentrations of neurotransmitters derived from Tyr and Trp were not significantly different with AA-MF or GMP-MF. Plasma serotonin levels of gut origin were higher in subjects with variant compared with classical PKU, and with GMP-MF compared with AA-MF in subjects with variant PKU. Metabolomics analysis identified higher levels of microbiome-derived compounds synthesized from Tyr, such as phenol sulfate, and higher levels of compounds synthesized from Trp in the kynurenine pathway, such as quinolinic acid, with ingestion of AA-MF compared with GMP-MF. CONCLUSIONS: The Tyr from AA-MF is less bioavailable due, in part, to greater degradation by intestinal microbes compared with the Tyr from prebiotic GMP-MF. Research is needed to understand how metabolism of Trp via the kynurenine pathway and changes in the intestinal microbiota affect health for individuals with PKU. This trial is registered at www.clinicaltrials.gov as NCT01428258.

Differential Effects of Dietary Fat Content and Protein Source on Bone Phenotype and Fatty Acid Oxidation in Female C57Bl/6 Mice.

BACKGROUND: Glycomacropeptide (GMP) is a 64-amino acid glycophosphopeptide released from κ-casein during cheesemaking that promotes satiety, reduces body fat, increases bone mass and infers prebiotic and anti-inflammatory effects. The impact of adiposity and gender on bone health is unclear. OBJECTIVE: To determine how feeding female mice diets providing 60% Fat Kcal (high-fat) or 13% Fat Kcal (control) with either GMP or casein as the protein source impacts: body composition, ex vivo fatty acid oxidation, bone (femoral) biomechanical performance, and the relationship between body composition and bone. METHODS: Weanling female C57Bl/6 mice were fed high-fat (60% Fat Kcal) or control diets (13% Fat Kcal) with GMP or casein from 3 to 32 weeks of age with assessment of body weight and food intake. Body composition was assessed by dual-energy X-ray absorptiometry (DXA). Fatty acid oxidation was measured in liver, muscle, and fat tissues using 14C-palmitate. Plasma concentrations of hormones and cytokines were determined. Bone biomechanical performance was assessed by the 3-point bending test. RESULTS: Female mice fed high-fat diets showed increased fatty acid oxidation capacity in both gastrocnemius muscle and brown adipose tissue compared to mice fed the control diets with a lower fat content. Despite increased fat mass in mice fed the high-fat diets, there was little evidence of glucose impairment or inflammation. Mice fed the high-fat diets had significantly greater total body bone mineral density (BMD), femoral BMD, and femoral cross-sectional area than mice fed the control diets. Femora of mice fed the high-fat diets had increased yield load and maximum load before fracture, consistent with greater bone strength, but reduced post-yield displacement or ductility, consistent with bone brittleness. Female mice fed a high-fat GMP diet displayed increased fat oxidation capacity in subcutaneous fat relative to mice fed the high-fat casein diet. Regardless of dietary fat content, GMP increased total body bone mineral content and femur length. The prebiotic properties of GMP may mediate the beneficial effects of GMP on bone. CONCLUSIONS: Female mice adapt to high-fat feeding by increasing oxidative capacity in muscle tissue and to a lesser extent brown adipose tissue. High-fat feeding in female mice leads to development of a bone phenotype where femora show increased BMD and are stronger, yet more brittle. The increased brittleness of bone was associated with increased body fat content due to high-fat feeding. In summary, high-fat feeding in female mice increases mineralization of bone, but negatively impacts bone quality resulting in brittle bones.

Glycomacropeptide is a prebiotic that reduces Desulfovibrio bacteria, increases cecal short-chain fatty acids, and is anti-inflammatory in mice.

Glycomacropeptide (GMP) is a 64-amino acid (AA) glycophosphopeptide with application to the nutritional management of phenylketonuria (PKU), obesity, and inflammatory bowel disease (IBD). GMP is a putative prebiotic based on extensive glycosylation with sialic acid, galactose, and galactosamine. Our objective was to determine the prebiotic properties of GMP by characterizing cecal and fecal microbiota populations, short-chain fatty acids (SCFA), and immune responses. Weanling PKU (Pah(enu2)) and wild-type (WT) C57Bl/6 mice were fed isoenergetic AA, GMP, or casein diets for 8 wk. The cecal content and feces were collected for microbial DNA extraction to perform 16S microbiota analysis by Ion Torrent PGM sequencing. SCFA were determined by gas chromatography, plasma cytokines via a Bio-Plex Pro assay, and splenocyte T cell populations by flow cytometry. Changes in cecal and fecal microbiota are primarily diet dependent. The GMP diet resulted in a reduction from 30-35 to 7% in Proteobacteria, genera Desulfovibrio, in both WT and PKU mice with genotype-dependent changes in Bacteroidetes or Firmicutes. Cecal concentrations of the SCFA acetate, propionate, and butyrate were increased with GMP. The percentage of stimulated spleen cells producing interferon-γ (IFN-γ) was significantly reduced in mice fed GMP compared with casein. In summary, plasma concentrations of IFN-γ, TNF-α, IL-1ß, and IL-2 were reduced in mice fed GMP. GMP is a prebiotic based on reduction in Desulfovibrio, increased SCFA, and lower indexes of inflammation compared with casein and AA diets in mice. Functional foods made with GMP may be beneficial in the management of PKU, obesity, and IBD.

Differential effects of low-phenylalanine protein sources on brain neurotransmitters and behavior in C57Bl/6-Pah(enu2) mice.

Phenylketonuria (PKU) is an inborn error of metabolism caused by a deficiency of the enzyme phenylalanine hydroxylase, which metabolizes phenylalanine (phe) to tyrosine. A low-phe diet plus amino acid (AA) formula is necessary to prevent cognitive impairment; glycomacropeptide (GMP) contains minimal phe and provides a palatable alternative to the AA formula. Our objective was to assess neurotransmitter concentrations in the brain and the behavioral phenotype of PKU mice (Pah(enu2) on the C57Bl/6 background) and how this is affected by low-phe protein sources. Wild type (WT) and PKU mice, both male and female, were fed high-phe casein, low-phe AA, or low-phe GMP diets between 3 and 18 weeks of age. Behavioral phenotype was assessed using the open field and marble burying tests, and brain neurotransmitter concentrations were measured using HPLC with electrochemical detection system. Data were analyzed by 3-way ANOVA with genotype, sex, and diet as the main treatment effects. Brain mass and the concentrations of catecholamines and serotonin were reduced in PKU mice compared to WT mice; the low-phe AA and GMP diets improved these parameters in PKU mice. Relative brain mass was increased in female PKU mice fed the GMP diet compared to the AA diet. PKU mice exhibited hyperactivity and impaired vertical exploration compared to their WT littermates during the open field test. Regardless of genotype or diet, female mice demonstrated increased vertical activity time and increased total ambulatory and horizontal activity counts compared with male mice. PKU mice fed the high-phe casein diet buried significantly fewer marbles than WT control mice fed casein; this was normalized in PKU mice fed the low-phe AA and GMP diets. In summary, C57Bl/6-Pah(enu2) mice showed an impaired behavioral phenotype and reduced brain neurotransmitter concentrations that were improved by the low-phe AA or GMP diets. These data support lifelong adherence to a low-phe diet for PKU.