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.

Skeletal myopathy in CKD: a comparison of adenine-induced nephropathy and 5/6 nephrectomy models in mice

Preclinical models of chronic kidney disease (CKD) are critical to investigate the mechanisms of disease and to evaluate novel therapeutics aimed to treat CKD-associated pathologies. The objective of the present study was to compare the adenine diet and 5/6 nephrectomy (5/6 Nx) models in mice. Male and female 10-week-old C57BL/6J mice (N=5-9/sex/group) were randomly allocated to CKD groups (0.2-0.15% adenine-supplemented diet or 5/6 Nx surgery) or corresponding control groups (casein diet or sham surgery). Glomerular filtration rate was reduced to a similar level in adenine and 5/6 Nx mice (adenine male: 81.1 ± 41.9 µL/min vs. 5/6 Nx male: 160 ± 80.9 µL/min, P=0.5875; adenine female: 112.9 ± 32.4 µL/min vs. 5/6 Nx female: 107.0 ± 45.7 µL/min, P=0.9995). Serum metabolomics analysis indicated that established uremic toxins were robustly elevated in both CKD models, although some differences were observed between CKD models (i.e. p-Cresol sulfate). Dysregulated phosphate homeostasis was observed in the adenine model only, whereas calcium homeostasis was disturbed in male mice with both models. Muscle mass and myofiber cross-sectional area of extensor digitorum longus and soleus muscles were ~18-24% smaller in male CKD mice regardless of model, but were not different in female CKD mice (P>0.05). Skeletal muscle mitochondrial respiratory function was significantly decreased (19-24%) in CKD mice in both models and sexes. These findings demonstrate that adenine diet and 5/6 Nx models of CKD have similar levels of renal dysfunction and skeletal myopathy, but had less mortality (P<0.05 for both sexes) compared with the 5/6 Nx model.

A Diet Based on Mesquite Seed Improves Atherogenic Index in Mouse Model

Objectives The main objective of this work is to evaluate the effect of a diet rich in antioxidants based on mesquite seed on the atherogenic index of the dislipidemia-prone C57BL/6J mouse. Methods Mesquite seed based diet with 18% of protein was prepared using flour of mesquite seed, corn and micronutrient mix. Additionally, a diet based on casein with 18% of protein was preparing using casein, corn flour, wheat bran and micronutrient mix. Both diets were processed by extrusion. Extruded diets were analyzed for proximal composition, flavonoids content (HPLC) and radical scavenging capacity. For mice biological assay development, a bioethically approved protocol was used. Four-week-old male C57BL/6J mice were used. One group of mice (n = 6) was fed a diet based on mesquite seed and a second group (n = 6) was fed a diet based on casein from week 4 to week 18 of life. Mice were maintained with free access to food and water. At the end of the experimental period, blood samples were taken for the determination of total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-c) using dry-chemistry enzymatic slide technology. The atherogenic index (AI) was calculated using the formula: AI = TC (mg/dL)/HDL-c (mg/dL). Results The caloric content of the diet based on mesquite seed and the diet based on casein were 373.1 and 365.1 kcal/100g, respectively. The energy intake of mice treated with mesquite and casein diet were 13.43 and 13.51 kcal/day, respectively. The phenolic compounds intake of mice treated with mesquite and casein diet were 8.3 and 1.9 mg/day, respectively. The atherogenic index in mice treated with a diet based on mesquite seed was 2.25 and for the mice treated with a diet based on casein was 3.31. Conclusions After 14 weeks of a diet based on mesquite seed C57BL/6J mice presented significantly lower levels of total cholesterol and atherogenic index than mice treated with a diet based on casein. Diet based on mesquite seed lead to higher intakes of phenolic compounds and antioxidants than a diet based on casein. The C57BL/6J mouse is a nicotinamide nucleotide transhydrogenase deficient model, which induces oxidative stress and insulin resistance. This condition increases the susceptibility to develops dyslipidemia, diabetes and obesity. A diet based on mesquite seed prevents dyslipidemia development in C57BL/6J. Funding Sources No funding.

Nutritional evaluation of cassava root meal fermented with rumen filtrate in rats.

The study reported herein evaluates with rats the nutritional quality of cassava root meal (CRM) fermented with rumen filtrate using caged layer waste (CLW), pig excreta (PE) and a 1:1 mix of CLW and PE respectively as sources of nitrogen. Some safety aspects regarding possible feed use were also investigated. Wistar rats were fed five purified diets viz; a basal diet (nitrogen free), a reference diet that contained casein and three test diets made of the enhanced CRM. Dietary treatments significantly influenced performance, biological indices and blood parameters (P<0.05). The performances of rats on the enhanced cassava diets were inferior 10 that of rats on casein diet. The level of serum urea and thiocyanate were significantly elevated for rats on the cassava root-based diets compared to the casein diet. Among the enhanced cassava test protein, Protein Efficiency Ratio (PER) values of 0.88, 0.57 and 0.62; Net Protein Ratio (NPR) values of 0.27, -0.15 and -0.03 and Biological Value (BV) of 55.04, 39.96 and 52.27 were obtained respectively for cassava enhanced with caged layer waste (CCLW), cassava enhanced with pig excreta (CPE) and cassava with 1.1 mix of caged layer waste and pig excreta (CCLPE). The result obtained from rats on CCLW was significantly better than the other enhanced cassava products.

Effects of Albumin Infusion on Serum Levels of Albumin, Proinflammatory Cytokines (TNF-α, IL-1, and IL-6), CRP, and MMP-8; Tissue Expression of EGRF, ERK1, ERK2, TGF-β, Collagen, and MMP-8; and Wound Healing in Sprague Dawley Rats

In this study, we sought to determine the roles of albumin in wound healing, which is infused both pre- and postoperatively in malnourished patients presenting with hypoalbuminemia. For the purposes of the study, we used 25 male Sprague Dawley rats of predetermined weight and age, which were initially maintained in a standard environment and fed the same diet for 7 days prior to being segregated into one of the following five groups: A, control, normal protein feed (20% casein); B, hypoalbuminemia, 25% rat albumin infusion prior to surgery; C, hypoalbuminemia, normal protein feed (20% casein); D, hypoalbuminemia, 25% rat albumin infusion after surgery; and E, hypoalbuminemia, low-protein feed (casein 2%). The animals in all five groups were subjected to four deep incisions in their dorsal muscle fascia. On days 1, 3, 5, and 7 after surgery, ELISA was used to determine serum levels of TNF-α, IL-1, IL-6, CRP, and MMP-8, whereas immunohistochemistry was used to determine the tissue expression of EGFR, ERK1, ERK2, TGF-β, collagen, and MMP-8. Significant reductions in serum levels of TNF-α, IL-1, and CRP were detected in the groups receiving albumin infusion and the high-casein diet (P<0.05). The administration of albumin and a high-casein diet also increased the tissue expression of EGFR, ERK1, ERK2, TGF-β, and collagen and decreased that of MMP-8 relative to the hypoalbuminemia control (P<0.05). We propose that the administration of albumin promoted NF-κB signaling which, in turn, induced the transduction and transcription of factors involved in wound healing. Albumin infusion and dietary proteins play vital roles in accelerating the wound healing process, as they can contribute to correcting the hypoalbuminemic state. These findings provide insights that will contribute to our understanding of wound healing, particularly in malnourished patients.

Adverse Effects of Autoclaved Diets on the Progression of Chronic Kidney Disease and Chronic Kidney Disease-Mineral Bone Disorder in Rats

Background: Autoclaving rodent diets is common in laboratory animals, but autoclaving increases the formation of dietary advanced glycation end-products (AGE). We studied the effect of autoclaved (AC) diet alone or in combination with a diet high in bioavailable phosphorus on biochemistries of chronic kidney disease-mineral and bone disorder (CKD-MBD), intestinal gene expression, and oxidative stress. Methods: Male CKD rats (Cy/+) and normal littermates were fed 1 of 3 diets: AC 0.7% phosphorus grain-based diet for 28 weeks (AC); AC diet for 17 weeks followed by non-autoclaved (Non-AC) 0.7% phosphorus casein diet until 28 weeks (AC + Casein); or Non-AC diet for 16 weeks followed by a Non-AC purified diet until 30 weeks (Non-AC + Casein). Results: AC diets contained ~3× higher AGEs and levels varied depending on the location within the autoclave. Rats fed the AC and AC + Casein diets had higher total AGEs and oxidative stress, irrespective of kidney function. Kidney function was more severely compromised in CKD rats fed AC or AC + Casein compared to Non-AC + Casein. There was a disease-by-diet interaction for plasma phosphorus, parathyroid hormone, and c-terminal fibroblast growth factor-23, driven by high values in the CKD rats fed the AC + Casein diet. Compared to Non-AC + Casein, AC and AC + Casein-fed groups had increased expression of receptor of AGEs and intestinal NADPH oxidase dual oxidase-2, independent of kidney function. Conclusions: Autoclaving rodent diets impacts the progression of CKD and CKD-MBD, highlighting the critical importance of standardizing diets in experiments.

Beneficial effects of linum usitatissimum L. on dyslipidemia, oxidative stress and inflammatory cytokines in hypercholesterolemic rats

Purpose This paper aims to study the effects of a diet supplemented with flaxseeds on dyslipidemia, oxidative stress and proinflammatory cytokines, in rats consuming a high-cholesterol diet. Design/methodology/approach Male Wistar rats (n = 30) weighing (250 ± 5 g) of which 10 were control and 20 were rendered hypercholesterolemic (HC) by feeding a diet enriched with 1% of cholesterol, for 15 days. After this phase, rats were divided into two groups; hypercholesterolemic group (HC) (n = 10), fed 20% casein diet enriched with 1% cholesterol; and hypercholesterolemic rats fed the same diet (n = 10), but additionally supplemented with flaxseeds (Linum usitatissimum) (Lu) powder, i.e. HC-Lu. Animals of the control group (n = 10) were fed the casein diet. All the animals were maintained on the respective diets for four weeks. Findings This study showed that in HC-Lu as compared to HC group, plasma total cholesterol, triacylglycerols and non-HDL cholesterol concentrations were respectively 2.4-, 1.5- and 3-fold lower. Also, the lipid peroxidation was reduced in red blood cells, organs (liver, heart and aorta) and lipoproteins (HDL2, HDL3 and VDL-LDL). A higher superoxide dismutase activity was observed in liver (+61%), heart (+62%) and aorta (+59%), whereas plasma proinflammatory cytokine (IL-1beta and IL-6) levels were decreased. Originality/value These results suggest that flaxseeds help to reduce hypercholesterolemia, oxidative stress and inflammation in patients with hypercholesterolemia.

Splenohepatomegaly in Protein-Malourished Mice Infected with Plasmodium Vinckei

Cellular and histopathological observations were made with light microscopy during the Plasmodium vinckeiinfection in protein-malnourished mice (fed on 2% casein diet and para-aminobenzoic acid deficient diet (PABA-DD).In the infected spleen, hyperplasia of the red as well as the white pulp was evident; active erythropoiesis and lymphopoiesis. Hyperemia and deposition of malarial pigments inside the macrophages. Cloudy swelling was noticed in the swollen hepatic cells of the uninfected liver due to protein deficiency. This lesion had developed to fatty infiltration in the infected protein-malnourished mice. These new and interesting changes represent the combined deleterious effect upon the host of infection and protein-malnutrition. Hypertrophy of the kupffer cells due to progressive phagocytosis of malarial pigments. In addition, focal necrosis surrounded by inflammatory cells was seen.

Nutritional evaluation of mixed wheat–faba bean pasta in growing rats: impact of protein source and drying temperature on protein digestibility and retention

This study aimed to evaluate the nutritional value of pasta enriched with legume or wheat gluten proteins and dried at varying temperature. A total of four isonitrogenous experimental diets were produced using gluten powder/wheat semolina (6/94, g/g) pasta and faba bean flour/wheat semolina (35/65, g/g) pasta dried at either 55°C (GLT and FLT, respectively) or 90°C (FVHT and GVHT, respectively). Experimental diets were fed to ten 1-month-old Wistar rats (body weight=176 (sem 15) g) for 21 d. Growth and nutritional, metabolic and inflammatory markers were measured and compared with an isonitrogenous casein diet (CD). The enrichment with faba bean increased the lysine, threonine and branched amino acids by 97, 23 and 10 %, respectively. Protein utilisation also increased by 75 % (P<0·01) in FLT in comparison to GLT diet, without any effect on the corrected faecal digestibility (P>0·05). Faba bean pasta diets' corrected protein digestibility and utilisation was only 3·5 and 9 %, respectively, lower than the CD. Growth rate, blood composition and muscle weights were not generally different with faba bean pasta diets compared with CD. Corrected protein digestibility was 3 % lower in GVHT than GLT, which may be associated with greater carboxymethyllysine. This study in growing rats clearly indicates improvement in growth performance of rats fed legume-enriched pasta diet compared with rats fed gluten–wheat pasta diet, regardless of pasta drying temperature. This means faba bean flour can be used to improve the protein quality and quantity of pasta.