The effect of casein glycomacropeptide versus free synthetic amino acids for early treatment of phenylketonuria in a mice model.

INTRODUCTION: Management of phenylketonuria (PKU) is mainly achieved through dietary control with limited intake of phenylalanine (Phe) from food, supplemented with low protein (LP) food and a mixture of free synthetic (FS) amino acids (AA) (FSAA). Casein glycomacropeptide (CGMP) is a natural peptide released in whey during cheese making by the action of the enzyme chymosin. Because CGMP in its pure form does not contain Phe, it is nutritionally suitable as a supplement in the diet for PKU when enriched with specific AAs. Lacprodan® CGMP-20 (= CGMP) used in this study contained only trace amounts of Phe due to minor presence of other proteins/peptides. OBJECTIVE: The aims were to address the following questions in a classical PKU mouse model: Study 1, off diet: Can pure CGMP or CGMP supplemented with Large Neutral Amino Acids (LNAA) as a supplement to normal diet significantly lower the content of Phe in the brain compared to a control group on normal diet, and does supplementation of selected LNAA results in significant lower brain Phe level?. Study 2, on diet: Does a combination of CGMP, essential (non-Phe) EAAs and LP diet, provide similar plasma and brain Phe levels, growth and behavioral skills as a formula which alone consist of FSAA, with a similar composition?. MATERIAL AND METHODS: 45 female mice homozygous for the Pahenu2 mutation were treated for 12 weeks in five different groups; G1(N-CGMP), fed on Normal (N) casein diet (75%) in combination with CGMP (25%); G2 (N-CGMP-LNAA), fed on Normal (N) casein diet (75%) in combination with CGMP (19,7%) and selected LNAA (5,3% Leu, Tyr and Trp); G3 (N), fed on normal casein diet (100%); G4 (CGMP-EAA-LP), fed on CGMP (70,4%) in combination with essential AA (19,6%) and LP diet; G5 (FSAA-LP), fed on FSAA (100%) and LP diet. The following parameters were measured during the treatment period: Plasma AA profiles including Phe and Tyr, growth, food and water intake and number of teeth cut. At the end of the treatment period, a body scan (fat and lean body mass) and a behavioral test (Barnes Maze) were performed. Finally, the brains were examined for content of Phe, Tyr, Trp, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and 5-hydroxyindole-acetic acid (5-HIAA), and the bone density and bone mineral content were determined by dual-energy x-ray absorptiometry. RESULTS: Study 1: Mice off diet supplemented with CGMP (G1 (N-CGMP)) or supplemented with CGMP in combination with LNAA (G2 (N-CGMP-LNAA)) had significantly lower Phe in plasma and in the brain compared to mice fed only casein (G3 (N)). Extra LNAA (Tyr, Trp and Leu) to CGMP did not have any significant impact on Phe levels in the plasma and brain, but an increase in serotonin was measured in the brain of G2 mice compared to G1. Study 2: PKU mice fed with mixture of CGMP and EAA as supplement to LP diet (G4 (CGMP-EAA-LP)) demonstrated lower plasma-Phe levels but similar brain- Phe levels and growth as mice fed on an almost identical combination of FSAA (G5 (FSAA-LP)). CONCLUSION: CGMP can be a relevant supplement for the treatment of PKU.

Oral D/L-3-Hydroxybutyrate Stimulates Cholecystokinin and Insulin Secretion and Slows Gastric Emptying in Healthy Males.

BACKGROUND: D-3-hydroxybutyrate (D-3-OHB) is a ketone body that serves as an alternative nutritional fuel but also as an important signaling metabolite. Oral ketone supplements containing D/L-3-OHB are becoming a popular approach to achieve ketosis. AIM: To explore the gut-derived effects of ketone supplements. METHODS: Eight healthy lean male volunteers were investigated on 2 separate occasions:An acetaminophen test was performed to evaluate gastric emptying and blood samples were obtained consecutively throughout the study period. RESULTS: We show that oral consumption of D/L-3-OHB stimulates cholecystokinin release (P = 0.02), elevates insulin (P = 0.03) and C-peptide (P < 0.001) concentrations, and slows gastric emptying (P = 0.01) compared with matched intravenous D/L-3-OHB administration. Measures of appetite and plasma concentrations of glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) were unaffected by interventions. CONCLUSION: Our findings show that D/L-3-OHB exert incretin effects and indicate luminal sensing in the gut endothelium. This adds to our understanding of ketones as signaling metabolites and displays the important difference between physiological ketosis and oral ketone supplements.

Sensitive determination of monoamine neurotransmitters, their main metabolites and precursor amino acids in different mouse brain components by liquid chromatography-electrospray tandem mass spectrometry after selective sample clean-up.

For the assessment of diets and supplements formulated for the treatment of phenylketonuria, a highly sensitive and selective method was developed and validated for the quantification of dopamine (DA), serotonin (5-HT), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA), phenylalanine, tyrosine and tryptophan in mouse cerebellum, brain stem, hypothalamus, parietal cortex, anterior piriform cortex and bulbus olfactorius. Samples were extracted by deproteinization with acetonitrile, and the extracts were cleaned up by strong anion exchange and weak cation exchange applied sequentially. The substances were detected by rapid liquid chromatography tandem mass spectrometry. Matrix components were largely removed by the clean-up, resulting in low matrix effects. The lower limits of quantification for an extracted tissue mass of 100 mg were 0.3, 0.3, 0.2 and 2 ng/g for DA, 5-HT, 5-HIAA and DOPAC, respectively. The mean true extraction recoveries were 80-102%. The relative intra-laboratory reproducibility standard deviations were generally <11% at concentrations of 20-1000 ng/g for DA, 5-HT, 5-HIAA and DOPAC and 7% at concentrations of 5-50 µg/g for the amino acids. This method was successfully used in a phenylketonuria mice study including nearly 300 brain tissue samples and for small sample masses (for example, 2 mg of bulbus olfactorius).