Metabolomic biomarkers in autism: identification of complex dysregulations of cellular bioenergetics.

Autism Spectrum Disorder (ASD or autism) is a phenotypically and etiologically heterogeneous condition. Identifying biomarkers of clinically significant metabolic subtypes of autism could improve understanding of its underlying pathophysiology and potentially lead to more targeted interventions. We hypothesized that the application of metabolite-based biomarker techniques using decision thresholds derived from quantitative measurements could identify autism-associated subpopulations. Metabolomic profiling was carried out in a case-control study of 499 autistic and 209 typically developing (TYP) children, ages 18-48 months, enrolled in the Children's Autism Metabolome Project (CAMP; ClinicalTrials.gov Identifier: NCT02548442). Fifty-four metabolites, associated with amino acid, organic acid, acylcarnitine and purine metabolism as well as microbiome-associated metabolites, were quantified using liquid chromatography-tandem mass spectrometry. Using quantitative thresholds, the concentrations of 4 metabolites and 149 ratios of metabolites were identified as biomarkers, each identifying subpopulations of 4.5-11% of the CAMP autistic population. A subset of 42 biomarkers could identify CAMP autistic individuals with 72% sensitivity and 90% specificity. Many participants were identified by several metabolic biomarkers. Using hierarchical clustering, 30 clusters of biomarkers were created based on participants' biomarker profiles. Metabolic changes associated with the clusters suggest that altered regulation of cellular metabolism, especially of mitochondrial bioenergetics, were common metabolic phenotypes in this cohort of autistic participants. Autism severity and cognitive and developmental impairment were associated with increased lactate, many lactate containing ratios, and the number of biomarker clusters a participant displayed. These studies provide evidence that metabolic phenotyping is feasible and that defined autistic subgroups can lead to enhanced understanding of the underlying pathophysiology and potentially suggest pathways for targeted metabolic treatments.

Glycomacropeptide Impacts Amylin-Mediated Satiety, Postprandial Markers of Glucose Homeostasis, and the Fecal Microbiome in Obese Postmenopausal Women.

BACKGROUND: Obesity with metabolic syndrome is highly prevalent and shortens lifespan. OBJECTIVES: In a dose-finding crossover study, we evaluated the effect of glycomacropeptide (GMP) on satiety, glucose homeostasis, amino acid concentrations, inflammation, and the fecal microbiome in 13 obese women. METHODS: Eligible women were ≤10 yr past menopause with a body mass index [BMI (in kg/m2)] of 28 to 35 and no underlying inflammatory condition affecting study outcomes. Participants consumed GMP supplements (15 g GMP + 10 g whey protein) twice daily for 1 wk and thrice daily for 1 wk, with a washout period between the 2 wk. Women completed a meal tolerance test (MTT) on day 1 (soy MTT) and day 7 (GMP MTT) of each week. During each test, subjects underwent measures of glucose homeostasis, satiety, cytokines, and the fecal microbiome compared with that of usual diet, and rated the acceptability of consuming GMP supplements. RESULTS: The mean ± SE age of the 13 women was 57 ± 1 yr, with a median of 8 yr (range: 3-9 yr) past menopause and a BMI of 30 (IQR: 29-32). GMP was highly acceptable to participants, permitting high adherence. Metabolic effects were similar for twice or thrice daily GMP supplementation. Glucose, insulin, and cytokine concentrations were no different. The postprandial area under the curve (AUC) for glucagon concentrations was significantly lower, and the insulin-glucagon ratio was significantly higher with GMP than that with the soy MTT. Postprandial AUC amylin concentration was significantly higher with GMP than that with the soy MTT and correlated with C-peptide (P < 0.001; R2 = 0.52) and greater satiety. Ingestion of GMP supplements twice daily reduced members of the genus Streptococcus (P = 0.009) and thrice daily consumption reduced overall α diversity. CONCLUSIONS: GMP is shown to increase amylin concentrations, improve glucose homeostasis, and alter the fecal microbiome. GMP can be a helpful nutritional supplement in obese postmenopausal women at risk for metabolic syndrome. Further investigation is warranted. This trial was registered at clinicaltrials.gov as NCT05551091.

A Metabolomics Approach to Screening for Autism Risk in the Children's Autism Metabolome Project.

Autism spectrum disorder (ASD) is biologically and behaviorally heterogeneous. Delayed diagnosis of ASD is common and problematic. The complexity of ASD and the low sensitivity of available screening tools are key factors in delayed diagnosis. Identification of biomarkers that reduce complexity through stratification into reliable subpopulations can assist in earlier diagnosis, provide insight into the biology of ASD, and potentially suggest targeted interventions. Quantitative metabolomic analysis was performed on plasma samples from 708 fasting children, aged 18 to 48 months, enrolled in the Children's Autism Metabolome Project (CAMP). The primary goal was to identify alterations in metabolism helpful in stratifying ASD subjects into subpopulations with shared metabolic phenotypes (i.e., metabotypes). Metabotypes associated with ASD were identified in a discovery set of 357 subjects. The reproducibility of the metabotypes was validated in an independent replication set of 351 CAMP subjects. Thirty-four candidate metabotypes that differentiated subsets of ASD from typically developing participants were identified with sensitivity of at least 5% and specificity greater than 95%. The 34 metabotypes formed six metabolic clusters based on ratios of either lactate or pyruvate, succinate, glycine, ornithine, 4-hydroxyproline, or α-ketoglutarate with other metabolites. Optimization of a subset of new and previously defined metabotypes into a screening battery resulted in 53% sensitivity (95% confidence interval [CI], 48%-57%) and 91% specificity (95% CI, 86%-94%). Thus, our metabolomic screening tool detects more than 50% of the autistic participants in the CAMP study. Further development of this metabolomic screening approach may facilitate earlier referral and diagnosis of ASD and, ultimately, more targeted treatments. LAY SUMMARY: Analysis of a selected set of metabolites in blood samples from children with autism and typically developing children identified reproducible differences in the metabolism of about half of the children with autism. Testing for these differences in blood samples can be used to help screen children as young as 18 months for risk of autism that, in turn, can facilitate earlier diagnoses. In addition, differences may lead to biological insights that produce more precise treatment options. We are exploring other blood-based molecules to determine if still a higher percentage of children with autism can be detected using this strategy. Autism Res 2020, 13: 1270-1285. © 2020 The Authors. Autism Research published by International Society for Autism Research published by Wiley Periodicals LLC.

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.

ThedaCare Nutrition Risk Screen Improves the Identification of Non-Intensive Care Unit Patients at Risk for Malnutrition Compared With the Nutrition Risk Screen 2002.

BACKGROUND: Identification of patients at risk for malnutrition is important for timely nutrition intervention to reduce morbidity and mortality. OBJECTIVE: The objective of this study was to compare the sensitivity and specificity of the Nutrition Risk Screen (NRS) 2002 and the ThedaCare NRS to identify patients at risk for malnutrition. METHODS: The NRS 2002 and ThedaCare NRS were administered to 594 patients, aged 63 ± 16 years (mean ± SD), in the non-intensive care unit hospital setting. Risk for malnutrition and malnutrition diagnosis were confirmed with the 6 malnutrition clinical characteristics defined by the Academy of Nutrition and Dietetics and the American Society for Parenteral and Enteral Nutrition and using the nutrition assessment that included the Nutrition Focused Physical Exam. Sensitivity, specificity, and κ coefficient were calculated. RESULTS: When compared with the NRS 2002, the ThedaCare NRS had higher sensitivity (98.8% vs 63.5%), indicating improved identification of patients at risk for malnutrition, but lower specificity (74.0% vs 93.4%), indicating that more patients at low risk for malnutrition were misclassified. ThedaCare NRS missed fewer patients at risk for malnutrition when compared with the NRS 2002. ThedaCare NRS had a higher κ coefficient when compared with the NRS 2002, indicating better agreement of results regardless of who administered the screen. The ThedaCare NRS required less time to complete when compared with the NRS 2002 (mean ± SE: ThedaCare, 17 ± 1 seconds; NRS 2002, 9 ± 1 minutes; P < .0001). CONCLUSION: The ThedaCare NRS improves the identification of patients at risk for malnutrition in the non-intensive care unit hospital setting. This trial was registered at www.clinicaltrials.gov as NCT02585245.

Sex differences in body composition and bone mineral density in phenylketonuria: A cross-sectional study.

BACKGROUND: Low bone mineral density (BMD) and subsequent skeletal fragility have emerged as a long-term complication of phenylketonuria (PKU). OBJECTIVE: To determine if there are differences in BMD and body composition between male and female participants with PKU. METHODS: From our randomized, crossover trial [1] of participants with early-treated PKU who consumed a low-phenylalanine (Phe) diet combined with amino acid medical foods (AA-MF) or glycomacropeptide medical foods (GMP-MF), a subset of 15 participants (6 males, 9 females, aged 15-50 y, 8 classical and 7 variant PKU) completed one dual energy X-ray absorptiometry (DXA) scan and 3-day food records after each dietary treatment. Participants reported lifelong compliance with AA-MF. In a crossover design, 8 participants (4 males, 4 females, aged 16-35 y) provided a 24-h urine collection after consuming AA-MF or GMP-MF for 1-3 weeks each. RESULTS: Male participants had significantly lower mean total body BMD Z-scores (means ± SE, males = - 0.9 ± 0.4; females, 0.2 ± 0.3; p = 0.01) and tended to have lower mean L1-4 spine and total femur BMD Z-scores compared to female participants. Only 50% percent of male participants had total body BMD Z-scores above - 1.0 compared to 100% of females (p = 0.06). Total femur Z-scores were negatively correlated with intake of AA-MF (r = - 0.58; p = 0.048). Males tended to consume more grams of protein equivalents per day from AA-MF (means ± SE, males: 67 ± 6 g, females: 52 ± 4 g; p = 0.057). Males and females demonstrated similar urinary excretion of renal net acid, magnesium and sulfate; males showed a trend for higher urinary calcium excretion compared to females (means ± SE, males: 339 ± 75 mg/d, females: 228 ± 69 mg/d; p = 0.13). Females had a greater percentage of total fat mass compared to males (means ± SE, males: 24.5 ± 4.8%, females: 36.5 ± 2.5%; p = 0.047). Mean appendicular lean mass index was similar between males and females. Male participants had low-normal lean mass based on the appendicular lean mass index. CONCLUSIONS: Males with PKU have lower BMD compared with females with PKU that may be related to higher intake of AA-MF and greater calcium excretion. The trial was registered at www.clinicaltrials.gov as NCT01428258.

Metabolomic Markers of Essential Fatty Acids, Carnitine, and Cholesterol Metabolism in Adults and Adolescents with Phenylketonuria.

Background: Individuals with phenylketonuria (PKU) have a risk of cognitive impairment and inflammation. Many follow a low-phenylalanine (low-Phe) diet devoid of animal protein in combination with medical foods (MFs). Objective: To assess lipid metabolism in participants with PKU consuming amino acid MFs (AA-MFs) or glycomacropeptide MFs (GMP-MFs), we conducted fatty acid and metabolomics analyses. Methods: We used subsets of fasting plasma and urine samples from our randomized crossover trial in which participants with early-treated classical and variant (milder) PKU consumed a low-Phe diet combined with AA-MFs or GMP-MFs for 3 wk each. Fatty acid profiles of red blood cell (RBC) membranes were determined for 25 adults (aged 18-49 y) with PKU and 143 control participants. Metabolomics analyses of plasma and urine samples were conducted by Metabolon for 9-10 adolescent and adult participants with PKU and for 15 control participants. Results: RBC fatty acid profiles were not significantly different with AA-MFs or GMP-MFs. PKU participants showed higher total n-6:n-3 (ω-6:ω-3) fatty acids (mean ± SD percentages of total fatty acids: AA-MF = 5.45% ± 1.07%; controls = 4.33%; P < 0.001) and lower docosahexaenoic acid (DHA; AA-MF = 3.21% ± 0.98%; controls = 3.70% ± 1.01%; P = 0.02) and eicosapentaenoic acid (AA-MF = 0.33% ± 0.12%; controls = 0.60% ± 0.43%; P < 0.001) in RBCs than did control participants. Despite higher carnitine intake from AA-MFs than GMP-MFs (mean ± SE intake: AA-MFs = 58.6 ± 5.3 mg/d; GMP-MFs = 0.3 ± 0.01 mg/d; P < 0.001), plasma concentrations of carnitine were similar and not different from those in the control group (AA-MF compared with GMP-MF, P = 0.73). AA-MFs resulted in higher urinary excretion of trimethylamine N-oxide (TMAO), which is synthesized by bacteria from carnitine, compared with GMP-MFs (mean ± SE scaled intensity-TMAO: AA-MFs = 1.2 ± 0.1, GMP-MFs = 0.9 ± 0.1; P = 0.005). Plasma deoxycarnitine was lower in PKU participants than in control participants, suggesting reduced carnitine biosynthesis in PKU (AA-MF = 0.9 ± 0.1; GMP-MF = 1.0 ± 0.1; controls = 1.3 ± 0.1; AA-MF compared with controls, P = 0.01; GMP-MF compared with controls, P = 0.04). Conclusions: Supplementation with DHA is needed in PKU. Carnitine supplementation of AA-MFs shows reduced bioavailability due, in part, to bacterial degradation to TMAO, whereas the bioavailability of carnitine is greater with prebiotic GMP-MFs. This trial was registered at www.clinicaltrials.gov as NCT01428258.

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.

Metabolomic Insights into the Nutritional Status of Adults and Adolescents with Phenylketonuria Consuming a Low-Phenylalanine Diet in Combination with Amino Acid and Glycomacropeptide Medical Foods.

BACKGROUND: Nutrient status in phenylketonuria (PKU) requires surveillance due to the restrictive low-Phe diet in combination with amino acid medical foods (AA-MF) or glycomacropeptide medical foods (GMP-MF). Micronutrient profiles of medical foods are diverse, and optimal micronutrient supplementation in PKU has not been established. METHODS: In a crossover design, 30 participants with PKU were randomized to consume AA-MF and Glytactin™ GMP-MF in combination with a low-Phe diet for 3 weeks each. Fasting venipunctures, medical food logs, and 3-day food records were obtained. Metabolomic analyses were completed in plasma and urine by Metabolon, Inc. RESULTS: The low-Phe diets in combination with AA-MF and GMP-MF were generally adequate based on Dietary Reference Intakes, clinical measures, and metabolomics. Without micronutrient supplementation of medical foods, >70% of participants would have inadequate intakes for 11 micronutrients. Despite micronutrient supplementation of medical foods, inadequate intakes of potassium in 93% of participants and choline in >40% and excessive intakes of sodium in >63% of participants and folic acid in >27% were observed. Sugar intake was excessive and provided 27% of energy. CONCLUSIONS: Nutrient status was similar with AA-MF and Glytactin GMP-MF. More research related to micronutrient supplementation of medical foods for the management of PKU is needed.

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 for nutritional management of phenylketonuria: a randomized, controlled, crossover trial.

BACKGROUND: To prevent cognitive impairment, phenylketonuria requires lifelong management of blood phenylalanine (Phe) concentration with a low-Phe diet. The diet restricts intake of Phe from natural proteins in combination with traditional amino acid medical foods (AA-MFs) or glycomacropeptide medical foods (GMP-MFs) that contain primarily intact protein and a small amount of Phe. OBJECTIVE: We investigated the efficacy and safety of a low-Phe diet combined with GMP-MFs or AA-MFs providing the same quantity of protein equivalents in free-living subjects with phenylketonuria. DESIGN: This 2-stage, randomized crossover trial included 30 early-treated phenylketonuria subjects (aged 15-49 y), 20 with classical and 10 with variant phenylketonuria. Subjects consumed, in random order for 3 wk each, their usual low-Phe diet combined with AA-MFs or GMP-MFs. The treatments were separated by a 3-wk washout with AA-MFs. Fasting plasma amino acid profiles, blood Phe concentrations, food records, and neuropsychological tests were obtained. RESULTS: The frequency of medical food intake was higher with GMP-MFs than with AA-MFs. Subjects rated GMP-MFs as more acceptable than AA-MFs and noted improved gastrointestinal symptoms and less hunger with GMP-MFs. ANCOVA indicated no significant mean ± SE increase in plasma Phe (62 ± 40 µmol/L, P = 0.136), despite a significant increase in Phe intake from GMP-MFs (88 ± 6 mg Phe/d, P = 0.026). AA-MFs decreased plasma Phe (-85 ± 40 µmol/L, P = 0.044) with stable Phe intake. Blood concentrations of Phe across time were not significantly different (AA-MFs = 444 ± 34 µmol/L, GMP-MFs = 497 ± 34 µmol/L), suggesting similar Phe control. Results of the Behavior Rating Inventory of Executive Function were not significantly different. CONCLUSIONS: GMP-MFs provide a safe and acceptable option for the nutritional management of phenylketonuria. The greater acceptability and fewer side effects noted with GMP-MFs than with AA-MFs may enhance dietary adherence for individuals with phenylketonuria. This trial was registered at www.clinicaltrials.gov as NCT01428258.

Clinical relevance of the discrepancy in phenylalanine concentrations analyzed using tandem mass spectrometry compared with ion-exchange chromatography in phenylketonuria.

INTRODUCTION: Metabolic control of phenylketonuria (PKU) and compliance with the low-phenylalanine (phe) diet are frequently assessed by measuring blood phe concentrations in dried blood spots (DBS) collected by patients instead of plasma phe concentrations. OBJECTIVE: Our objective was to investigate the difference in blood phe concentrations in DBS collected by subjects and analyzed using either a validated newborn screening tandem mass spectrometry (MS/MS) protocol or ion-exchange chromatography (IEC) compared to plasma phe concentrations obtained simultaneously and analyzed using IEC. DESIGN: Three to four fasting blood samples were obtained from 29 subjects with PKU, ages 15-49 years. Capillary blood was spotted on filter paper by each subject and the DBS analyzed using both MS/MS and IEC. Plasma was isolated from venous blood and analyzed using IEC. RESULTS: Blood phe concentrations in DBS analyzed using MS/MS are 28% ± 1% (n = 110, p < 0.0001) lower than plasma phe concentrations analyzed using IEC resulting in a blood phe concentration of 514 ± 23 µmol/L and a plasma phe concentration of 731 ± 32 µmol/L (mean ± SEM). This discrepancy is larger when plasma phe is > 600 µmol/L. Due to the large variability across subjects of 13.2%, a calibration factor to adjust blood phe concentrations is not recommended. Analysis of DBS using IEC reduced the discrepancy to 15 ± 2% lower phe concentrations compared to plasma analyzed using IEC (n = 38, p = 0.0001). This suggests that a major contributor to the discrepancy in phe concentrations is the analytical method. CONCLUSION: Use of DBS analyzed using MS/MS to monitor blood phe concentrations in individuals with PKU yields significantly lower phe levels compared to plasma phe levels analyzed using IEC. Optimization of current testing methodologies for measuring phe in DBS, along with patient education regarding the appropriate technique for spotting blood on filter paper is needed to improve the accuracy of using DBS to measure phe concentrations in PKU management.

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.

Animal models of gastrointestinal and liver diseases. Animal models of infant short bowel syndrome: translational relevance and challenges.

Intestinal failure (IF), due to short bowel syndrome (SBS), results from surgical resection of a major portion of the intestine, leading to reduced nutrient absorption and need for parenteral nutrition (PN). The incidence is highest in infants and relates to preterm birth, necrotizing enterocolitis, atresia, gastroschisis, volvulus, and aganglionosis. Patient outcomes have improved, but there is a need to develop new therapies for SBS and to understand intestinal adaptation after different diseases, resection types, and nutritional and pharmacological interventions. Animal studies are needed to carefully evaluate the cellular mechanisms, safety, and translational relevance of new procedures. Distal intestinal resection, without a functioning colon, results in the most severe complications and adaptation may depend on the age at resection (preterm, term, young, adult). Clinically relevant therapies have recently been suggested from studies in preterm and term PN-dependent SBS piglets, with or without a functional colon. Studies in rats and mice have specifically addressed the fundamental physiological processes underlying adaptation at the cellular level, such as regulation of mucosal proliferation, apoptosis, transport, and digestive enzyme expression, and easily allow exogenous or genetic manipulation of growth factors and their receptors (e.g., glucagon-like peptide 2, growth hormone, insulin-like growth factor 1, epidermal growth factor, keratinocyte growth factor). The greater size of rats, and especially young pigs, is an advantage for testing surgical procedures and nutritional interventions (e.g., PN, milk diets, long-/short-chain lipids, pre- and probiotics). Conversely, newborn pigs (preterm or term) and weanling rats provide better insights into the developmental aspects of treatment for SBS in infants owing to their immature intestines. The review shows that a balance among practical, economical, experimental, and ethical constraints will determine the choice of SBS model for each clinical or basic research question.

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.

Advances in the nutritional and pharmacological management of phenylketonuria.

PURPOSE OF REVIEW: The purpose is to discuss advances in the nutritional and pharmacological management of phenylketonuria (PKU). RECENT FINDINGS: Glycomacropeptide (GMP), a whey protein produced during cheese production, is a low-phenylalanine (phe) intact protein that represents a new dietary alternative to synthetic amino acids for people with PKU. Skeletal fragility is a long-term complication of PKU that based on murine research, appears to result from both genetic and nutritional factors. Skeletal fragility in murine PKU is attenuated with the GMP diet, compared with an amino acid diet, allowing greater radial bone growth. Pharmacologic therapy with tetrahydrobiopterin, acting as a molecular chaperone for phenylalanine hydroxylase, increases tolerance to dietary phe in some individuals. Large neutral amino acids inhibit phe transport across the intestinal mucosa and blood-brain barrier, and are most effective for individuals unable to comply with the low-phe diet. SUMMARY: Although a low-phe synthetic amino acid diet remains the mainstay of PKU management, new nutritional and pharmacological treatment options offer alternative approaches to maintain lifelong low phe concentrations. GMP medical foods provide an alternative to amino acid formula that may improve bone health, and tetrahydrobiopterin permits some individuals with PKU to increase tolerance to dietary phe. Further research is needed to characterize the long-term efficacy of these new approaches for PKU management.