Negative effects of a low-quality protein diet on wound healing via modulation of the MMP2 activity in rats

Protein malnutrition is largely associated with a delay or failure of the healing process. However, the effect of dietary protein quality on wound healing is largely unknown. This study aimed to reveal the effect of dietary protein quality on wound healing and elucidate the regulatory mechanisms in a rat model of full-thickness cutaneous wounds. Rats were fed a normal diet for a week, and then they were divided into three groups that were fed the following diet for the experimental period: casein diet, gluten diet and gluten + lysine diet. The gluten diet significantly decreased body weight and wound healing compared with the casein diet, but this effect was reversed by supplementation with lysine. The numbers of leukocytes were significantly higher in the skin of the gluten group than those in the casein group. The wounded skin tissues of the gluten group showed lower amounts of collagen deposition compared with that in the casein group. Our results also showed that both matrix metalloproteinase (MMP) 2 activity and MMP14 mRNA levels were significantly increased in the skin of the gluten group, compared with the casein group. In summary, this study suggests low-quality protein diets have negative effects on wound healing via modulation of MMP2 activity in rats.     

Physiological casein and gluten protein requirements of growing rats

Using mounting casein and wheat gluten protein values (0-40%) in the animals' diet, the optimum and minimum physiological daily doses were determined in 49-day-old growing rats from changes in their body water, body nitrogen and protein intake. The optimum physiological doses were identical with the peak of linearity of the given parameters, which coincided with a 15% casein protein and a 20% gluten protein concentration in the diet. This was also confirmed by the maximum body amino acid values, which were found in animals given a 15% casein or 20% gluten protein diet. It was further confirmed by the finding of significantly elevated alanine aminotransferase and aspartate aminotransferase activity in the liver of animals with a higher intake of the above protein sources. The minimum physiological dose of the given protein was determined from the equations of the regression curves in the presence of zero changes in the body nitrogen or body water content. The optimum physiological daily doses of casein and wheat gluten protein were 3.25 g and 4.05 g respectively. The minimum physiological daily doses of casein protein were 268 mg (from body nitrogen changes) and 371 mg (from body water changes) and the minimum physiological daily doses of gluten protein were 892 mg (from body nitrogen changes) and 1,000 mg (from body water changes). The above indicators demonstrate, in the presence of higher and high dietary concentrations, that an intake of the given proteins over and above the optimum physiological daily dose is at the very least uneconomical (gluten), if not harmful (casein), making this a highly topical problem for further study.     

Determination of physiological casein and wheat gluten protein requirements of adult rats aged 75-89 days

Mounting doses of casein and wheat gluten protein (from 0% to 40% in the diet) were given to adult male rats aged 75-89 days for 14 days. The optimum physiological daily dose, determined from changes in body nitrogen, body water and body weight, was 2.76 g/d for casein protein (corresponding to a 10% casein protein diet) and 3.67 g/d for wheat gluten protein (corresponding to a 15% gluten protein diet). Determined from body weight changes, the daily maintaining dose of casein or wheat gluten protein was 1.054 and 1.511 mg/d respectively, from body nitrogen changes 970 and 1.514 mg/d and from body water changes 1.124 and 1.637 mg/d. Compared with newly weaned animals aged 35-49 days, the optimum physiological daily dietary protein doses for adult animals fall, while the maintaining doses rise.     

The provocation test in children with cow-milk protein and gluten intolerance: evaluation of the clinical response and lesions in the mucous membrane of the small intestine

Provocation test (re-introduction of the noxious protein) was carried out in two groups of patients: (a) with intolerance to the cow-milk proteins (41 children) treated with milk-free diet for 6-24 months, and (b) with gluten intolerance (26 children) treated with gluten-free diet for 6-36 months. The following parameters were compared: type and frequency of the clinical symptoms seen in these patients prior to the introduction of allergen-free diet. Moreover, the type of observed morphological changes in the small intestine mucosa following provocation test were analysed in the groups of 7 patients. A two-year elimination of milk from the diet produces milk tolerance in about 61% patients; clinical symptoms in the remaining children are diversified. Re-introduction of gluten with the diet (provocation test) produces recurrence of gluten intolerance in 96% of children treated with gluten-free diet for 2-3 years. Recurrence of the disease was accompanied by the atrophy of the intestinal villi.     

Influence of Protein Source on Growth-depressing Effect of Excess Amino Acids in Young Rats

The influence of protein quality on the growth-depressing effect of excessive amount of 12 individual essential and semiessential amino acids was examined. Growing rats were fed for 3 weeks diets containing either 10.5% egg albumin or 11.6% wheat gluten (equivalent to the protein content of a 10% casein diet) supplemented with 5% of each of the l-amino acids. In general, the pattern of growth depression produced by the addition of excess amino acids to the egg albumin or the wheat gluten diet was similar to that of the case of casein diet obtained previously under the same experimental conditions. However, the extent of these effects was dependent not only upon the kind of amino acid supplemented with but also upon the source of protein used, and the depressing effect of each of excess amino acids added to the wheat gluten diet was usually severer than those added to casein and egg albumin diets. No evidence was noted of any striking changes in the liver protein and nucleic acid concentrations by either diets, but total liver protein, RNA and DNA contents were decreased in some amino acid groups of the egg albumin diet and in all amino acid groups of the wheat gluten diet except the lysine addition. The free amino acid level in plasma generally showed extreme elevation for the amino acid supplemented in excess in the diet, and in most cases the extent of the elevation was correlated with the growth depression.     

Effects of adding dietary lysine to a low gluten diet on the brain protein synthesis rate in aged rats

The purpose of this study was to find whether the addition of dietary lysine affected the rate of brain protein synthesis in aged rats fed on a gluten diet. Experiments were done on two groups of aged rats (30 wk) given the diets containing 5% gluten or 5% gluten + 0.3% lysine for 10 d. The fractional rates of protein synthesis in brain, liver, and kidney increased with an addition of dietary lysine. In brain, liver, and kidney, the RNA activity [g protein synthesized/(g RNA x d)] was significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg RNA/g protein) was not related to the fractional rate of protein synthesis in any organ. The results suggest that the addition of the limiting amino acid for the low quality protein elevates the rate of protein synthesis in the brain of aged rats, and that RNA activity is at least partly related to the fractional rate of brain protein synthesis.     

Design of azidoproline containing gluten peptides to suppress CD4+ T-cell responses associated with celiac disease

Celiac disease is an intestinal disease caused by intolerance for gluten, a common protein in food. A life-long gluten-free diet is the only available treatment. As it is well established that the interaction between proline-rich gluten derived peptides and the human HLA-DQ2 molecules induces immune responses that lead to disease development, we have now designed a series of gluten peptides in which proline residues were replaced by azidoprolines. These peptides were found to bind to HLA-DQ2 with an affinity similar to that of the natural gluten peptide. Moreover, some of these peptides were found to be non-immunogenic and block gluten induced immune responses. These can thus serve as lead compounds for the development of HLA-DQ2 blocker peptides.     

On the Relationship between Food Composition and Serine Dehydrase Activity in Rat Liver

Effects of the change of dietary protein on serine dehydrase activity in rat liver have been studied, using egg albumin, casein, rice protein, and wheat gluten as protein source. At 35% of dietary protein level, the activity induced by egg albumin and casein diets were higher than those by rice protein and wheat gluten diets. Parallel relation was observed between the enzyme activity and the protein intake. These results suggest that the dietary induction of this enzyme are based on the protein intake, which reflects the nutritional quality of dietary protein, rather than merely on the dietary protein level.

The contribution of individual amino acid for the enzyme induction by the egg albumin diet at 35% level was investigated, and it was concluded that this enzyme induction is dependent not on a specific amino acid but on the combined effect of each amino acid.     

Protein malnutrition and response of pancreatic acinar cells to stimulation by cholecystokinin

Pancreatic lobules were isolated from 2 groups of male Wistar rats after 23 days of diet. A control group (C) fed on a 20% protein diet (16% gluten + 4% casein) and an experimental group (E) on a 5% protein diet (4% gluten + 1% casein). After isolation, lobules were preincubated 10 min with 10 muCi [3H]-leucine, washed, then incubate within Krebs Ringer bicarbonate Hepes. Basal secretion, then stimulated secretion (50 pM of cholecystokinin (CCK] of radioactive and non-radioactive protein and amylase outputs were measured. During basal secretion, in (E) group, lobules secreted more proteins than (C) one, the same outputs of amylase and radioactive protein were observed in both groups. The stimulated secretion by CCK increased the outputs of non-radioactive protein and amylase of lobules (T) (2-3 fold), but was without effect on lobule (E) outputs. Therefore, a low-protein diet involved a decrease of CCK sensibility on acinar cells, this fact might be mediated by a decreasing number and/or affinity of their CCK receptors.     

Age dependence of the utilization of different quality proteins in animal experiments.

Male rats aged 45, 85, 145 and 270 days (daily body mass increments on an optimal diet containing casein were 6.73, 2.88, 0.53 and 0.31 g respectively) were fed 15 days ad libitum on a diet with a nutrient content physiological for their age, in which the protein source was milk casein (ratio of essential to nonessential amino acids E/N = 0.79, compensation coefficient K = 14) or wheat gluten (E/N = 0.30, K = -8). In the case of gluten, net protein utilization (NPU) fell markedly in rapidly growing animals aged 45 and 85 days (33 and 30% more than with casein), indicating that without essential amino acid compensation, gluten is inadequate for animals of this age, whose organism requires fully ensured proteosynthesis for growth and development. In adolescence and adulthood (145 and 270 days), the utilization of proteins is not dependent on their quality (the decrease in NPU 13 and 12%--is nonsignificant). That means that a smaller amount of essential amino acids, including the limiting amino acid in uncompensated protein, is sufficient for the maintenance and renewal of organs and tissues, i.e. for proteosynthesis. The activation of gluconeogenesis (phosphoenolpyruvate carboxykinase activity in the liver) after the intake of plant protein confirms the effect of proteins on catabolic processes.     

Studies on limiting essential amino acids in grieves as source of dietary protein for rainbow trout (oncorhynchus mykiss)

Diets were computed to contain equal concentrations of digestible crude protein either of wheat gluten (diet 1) or of grieves (diets 2–8). Per kg dry diet, 41 g crystalline amino acids were supplemented. All diets contained at least 1.2 g Lys per MJ digestible energy (DE). In diet 2, ratios of Met + Cys, Trp, Leu, Ile and Phe to Lys were about equal to those in diet 1. In each of diets 3–7, one of the respective amino acids, in diet 8 all five were replaced by Glu in the supplemented mixture of amino acids.

Each diet was fed to triplciate groups of 20 trout during a trial lasting 66 days. Trout fed the diet containing wheat gluten consumed more dry matter and showed higher growth rates as well as higher protein contents in their gained body mass than trout fed diets based on grieves. Supplementing Met plus Trp significantly improved dry matter intake, growth rate and protein content of gain, though not to the level of trout fed the wheat gluten diet, whereas Leu, Ile and Phe showed no such effect. When grieves were not supplemented with both Met and Trp, gain in body mass contained significantly more lipids. DE required per kg gain by trout fed wheat gluten, grieves + Met + Trp or grieves without supplementation of Met and Trp was 20.1, 21.2 and 29.9 MJ, respectively.     

Regulation of Amino Acid Intake in the Rat: Lysine Preference with Gluten Diets of Varying Lysine Content

Weanling rats were offered a choice of two diets, varying only in lysine content. The preference test was performed between a 20% gluten diet supplemented with 0.5% lysine HCl and a lysine-deficient amino acid mixture, 20% gluten or 20% gluten plus 0.2% lysine HCl diet, and was done between a 20% gluten diet and a 20% gluten diet containing 3, 5 or 7% lysine · HCl. Weight gain and food intake were not significantly different among all the self-selecting groups, and these values were almost the same as those in rats fed only the 20% gluten supplemented 0.5% lysine · HCl diet which obtained maximal growth. The lysine intake of the combination groups ranged from 67 to 240 mg per day (0.62-2.35% of consumed food). It was demonstrated that rats exhibit a definite ability to regulate lysine intake, and they select sufficient lysine to meet their requirements for this amino acid. The self-selection technique may be useful as a method to determine the requirements for amino acids.     

Humoral response to wheat protein in patients with coeliac disease and enteropathy associated T cell lymphoma.

Features that might distinguish uncomplicated coeliac disease from enteropathy associated T cell lymphoma were investigated. Of 76 patients with coeliac disease, 71 (93%) had raised levels of alpha gliadin antibody and all responded clinically and histologically to treatment with a gluten free diet. In contrast, none of 16 patients with enteropathy associated T cell lymphoma had raised levels of alpha gliadin antibody, and treatment with a gluten free diet resulted in histological improvement in one and transient clinical improvement in six patients. The ratio of women to men was 2.2:1 in the group with coeliac disease and 1:1.6 in the patients with enteropathy associated T cell lymphoma. Thus patients with enteropathy associated T cell lymphoma do not display a humoral immune response to wheat protein (alpha gliadin), rarely respond to a gluten free diet, and are often men. Patients with uncomplicated coeliac disease usually have raised levels of alpha gliadin antibody, always respond to a gluten free diet, and are frequently women. These findings suggest the presence of two separate forms of enteropathy: one is benign and sensitive to wheat protein whereas the other runs a malignant course.     

Creation of the first ultra‐low gluten barley (Hordeum vulgare L.) for coeliac and gluten‐intolerant populations

Coeliac disease is a well‐defined condition that is estimated to affect approximately 1% of the population worldwide. Noncoeliac gluten sensitivity is a condition that is less well defined, but is estimated to affect up to 10% of the population, and is often self‐diagnosed. At present, the only remedy for both conditions is a lifelong gluten‐free diet. A gluten‐free diet is often expensive, high in fat and low in fibre, which in themselves can lead to adverse health outcomes. Thus, there is an opportunity to use novel plant breeding strategies to develop alternative gluten‐free grains. In this work, we describe the breeding and characterization of a novel ultra‐low gluten (ULG) barley variety in which the hordein (gluten) content was reduced to below 5 ppm. This was achieved using traditional breeding strategies to combine three recessive alleles, which act independently of each other to lower the hordein content in the parental varieties. The grain of the initial variety was shrunken compared to wild‐type barleys. We implemented a breeding strategy to improve the grain size to near wild‐type levels and demonstrated that the grains can be malted and brewed successfully. The ULG barley has the potential to provide novel healthy foods and beverages for those who require a gluten‐free diet.     

Comparative study of the effects of 2 types of protein-energy malnutrition followed by a balanced refeeding, on the lipase, phospholipase A2 and amylase activities of the pancreas and pancreatic juice in the growing rat

The intake of 2 p. 100 casein diet as only protein source decreased overall phospholipase A2 activity. Amylase activity was more affected than lipase one. The effects of intake of 5 p. 100 gluten diet as only protein source were less important than 2 p. 100 casein diet. Refeeding on 20 p. 100 or 15 p. 100 casein diet caused a considerable increase of phospholipase A2, lipase and amylase activities.     

A Food-Grade Enzyme Preparation with Modest Gluten Detoxification Properties

Background and Aims

Celiac sprue is a life-long disease characterized by an intestinal inflammatory response to dietary gluten. A gluten-free diet is an effective treatment for most patients, but accidental ingestion of gluten is common, leading to incomplete recovery or relapse. Food-grade proteases capable of detoxifying moderate quantities of dietary gluten could mitigate this problem.

Methods

We evaluated the gluten detoxification properties of two food-grade enzymes, aspergillopepsin (ASP) from Aspergillus niger and dipeptidyl peptidase IV (DPPIV) from Aspergillus oryzae. The ability of each enzyme to hydrolyze gluten was tested against synthetic gluten peptides, a recombinant gluten protein, and simulated gastric digests of whole gluten and whole-wheat bread. Reaction products were analyzed by mass spectrometry, HPLC, ELISA with a monoclonal antibody that recognizes an immunodominant gluten epitope, and a T cell proliferation assay.

Results

ASP markedly enhanced gluten digestion relative to pepsin, and cleaved recombinant α2-gliadin at multiple sites in a non-specific manner. When used alone, neither ASP nor DPPIV efficiently cleaved synthetic immunotoxic gluten peptides. This lack of specificity for gluten was especially evident in the presence of casein, a competing dietary protein. However, supplementation of ASP with DPPIV enabled detoxification of moderate amounts of gluten in the presence of excess casein and in whole-wheat bread. ASP was also effective at enhancing the gluten-detoxifying efficacy of cysteine endoprotease EP-B2 under simulated gastric conditions.

Conclusions

Clinical studies are warranted to evaluate whether a fixed dose ratio combination of ASP and DPPIV can provide near-term relief for celiac patients suffering from inadvertent gluten exposure. Due to its markedly greater hydrolytic activity against gluten than endogenous pepsin, food-grade ASP may also augment the activity of therapeutically relevant doses of glutenases such as EP-B2 and certain prolyl endopeptidases.     

Comparative study of the effects of 2 types of protein-energy malnutrition followed by a balanced realimentation on trypsin and chymotrypsin activity of the pancreas and the pancreatic juice of the rat

The intake of 2 p. 100 casein or 5 p. 100 gluten diet as only protein source caused overall protein synthesis and immediately stopped the rat growth. Trypsin activity was less affected than chymotrypsin activity in pancreas. Refeeding on 20 p. 100 or 15 p. 100 casein diet caused a considerable increase of trypsin and chymotrypsin activities during the first week.     

Effects of dietary protein of proso millet on liver injury induced by D-galactosamine in rats

In this paper, we examined the effects of dietary protein from proso millet on liver injury induced by D-galactosamine or carbon tetrachloride in rats using serum enzyme activities as indices. D-galactosamine-induced elevations of serum activities of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase were significantly suppressed by feeding the diet containing 20% protein of proso millet for 14 days as compared with those of rats fed a 20% casein diet, but not in the case of carbon tetrachloride. The results showed that proso millet protein is effective at lower dietary protein levels than that of dietary gluten reported previously. Therefore, the findings reported here may suggest that proso millet protein is considered to be another preventive food for liver injury.     

Apparent digestion coefficients for dry matter, protein and essential amino acids in terrestrial ingredients for Pacific shrimp Litopenaeus vannamei (Decapoda: Penaeidae)

Protein quality mainly depends on the essential amino acid (EAA) profile, but also on its bioavailability, because EAA digestibility is generally lower than the analyzed amounts. This information is needed in the aquaculture industry for aquafeed formulation. For this purpose, the apparent digestibility coefficients of dry matter, protein, and essential amino acids of eight feedstuffs of terrestrial origin were determined for the juvenile whiteleg shrimp Litopenaeus vannamei (15-19 g), using 1% chromic oxide as an inert marker. A reference diet was formulated and produced in the laboratory. Eight experimental diets were prepared each with 30% of one of the experimental ingredients added to the reference diet: casein, porcine byproduct meal poultry byproduct meal, corn meal, wheat gluten meal, soybean paste, sorghum meal, and wheat meal. The experiment consisted of a single-factor, completely randomized design with three replicates per treatment. Samples of ingredients, diets and feces were analyzed for nitrogen and amino acids. For amino acid assay, we used reverse-phase high performance liquid chromatography. To avoid partial loss of methionine and cystine, samples of ingredients, diets, and feces were oxidized with performic acid to methionine sulfone and cysteic acid prior to acid hydrolysis. The apparent dry matter and protein digestive utilization coefficients varied from 68% to 109% and from 70% to 103%, respectively. Apparent digestibility of protein for casein, soy paste, wheat meal and wheat gluten were very high (over 90%), corn gluten and poultry byproducts meal showed high protein digestibility (over 80%), but porcine byproducts meal and sorghum meal had low digestibility (76% and 70%, respectively). There was a reasonable, but not total, correspondence between apparent protein digestibility and average essential amino acid digestibility coefficients, except for arginine in corn gluten, phenylalanine and leucine in sorghum meal, phenylalanine in soy paste and lysine in wheat meal and poultry by-product meal. The most digestible feed ingredients for whiteleg shrimp were: wheat gluten, wheat meal and soy paste; poultry byproduct meal and corn gluten were less digestible and the lowest digestibility occurred in porcine byproduct meal and sorghum meal. Feedstuffs exhibited great variability in dry matter, protein and amino acid digestive utilization coefficients, which should be considered when formulating shrimp feeds.     

Effect of adding dietary L-lysine, L-threonine and L-methionine to a low gluten diet on urea synthesis in rats

Summary. We have shown that urinary urea excretion increased in rats fed a low quality protein. The purpose of present study was to determine whether an addition of dietary limiting amino acids affected urea synthesis in rats fed a low gluten diet. Experiments were done on three groups of rats given diets containing 10% gluten, 10% gluten+0.5% L-lysine or 10% gluten+0.5% L-lysine, 0.2% L-threonine and 0.2% L-methionine for 10d. The urinary excretion of urea, and the liver concentrations of serine and ornithine decreased with the addition of dietary L-lysine, L-threonine and L-methionine. The fractional and absolute rates of protein synthesis in tissues increased with the treatment of limiting amino acids. The activities of hepatic urea-cycle enzymes was not related to the urea excretion. These results suggest that the addition of limiting amino acids for the low gluten diet controls the protein synthesis in tissues and hepatic ornithine and decline urea synthesis.