Application of total error concept in the analytical method validation for the assay of essential amino acids by precolumn derivatization

A pre-Ultraperformance Liquid Chromatography (UPLC) column derivatization procedure was developed for thesimultaneous quantification of essential amino acids (EAAs) in the solid oral dosage pharmaceutical formulation.This analytical procedure has been validated with the help of the concept of total error. The total error approach (thecombination of systematic and random error) is a decision-making tool for ensuring the performance of the method.Fluorenylmethyloxycarbonyl chloride was used as a derivatization reagent. The amino acid derivatives were separatedon a C18 column (internal diameter 2.1 × 100 mm, 1.6 µm) by gradient elution with 0.1% trifluoroacetic acid andacetonitrile:water (90:10, v/v), respectively, as mobile phase A and B. About 10 EAAs could be detected at 265 nm in35 minutes with a flow rate of 0.25 ml/min. The linearity range of each amino acid was between 0.1 and 1.0 mg/ml.The accuracy and risk profiles were considered acceptable across the range. The precolumn derivatization procedureand the concept of the validation of total error could be used as an appropriate strategy to demonstrate the suitabilityof the analytical procedure for the separation and evaluation of EAA in solid oral dosage formulations.

Reducción de la proteína bruta en la dieta de pollos criollos de engorde en un sistema semi-intensivo / Reduction of the crude protein in the diet of creole broilers in a semi-intensive system

RESUMEN Objetivo. Evaluar la reducción de los niveles de proteína bruta sin alterar la relación de aminoácidos esenciales en pollos de engorde criollos de uno a 42 días de edad. Materiales y métodos. Se realizaron dos ensayos que comprendieron la fase inicial (un a 21 días) y crecimiento (22 a 42 días). Se utilizaron 630 pollos de la línea Redbro de un día de edad, distribuidos en un diseño completamente al azar, con cinco tratamientos y seis repeticiones de 21 aves cada una, totalizando 30 unidades experimentales distribuidas en corrales con abrigo. Los niveles reducidos de la proteína bruta para la fase inicial fueron: 21.5; 21.0; 20.5; 20.0 y 19.5% y para la fase de crecimiento: 19.0; 18.5; 18.0; 17.5 y 17.0%. En todos los tratamientos se mantuvo la proporción mínima entre aminoácidos esenciales y lisina. Las características de desempeño evaluadas fueron: ganancia de peso, consumo de ración y conversión alimenticia. Resultados. No hubo diferencia (p>0.05) en la reducción de proteína bruta para ninguna de las variables en todas las fases, excepto para conversión alimenticia (p<0.05) en la fase de crecimiento que mejoró con 18.2% de proteína bruta. Conclusiones. Los niveles de proteína bruta para pollos criollos de engorde de la línea Redbro, pueden ser reducidos a 19.5 y 18.2% para la fases inicial y de crecimiento, respectivamente, siempre cuando se mantengan las relaciones adecuadas de aminoácidos con lisina digestible y se atiendan las exigencias de los aminoácidos limitantes.

ABSTRACT Objective . To evaluate the reduction of crude protein levels, without altering the ratio of essential amino acids, of free-range chickens, of one at 42 days old. Materials and methods. Two trials were performed comprising the initial phase (one to 21 days) and growth (22 to 42 days). A total of 630 chicks of the Redbro line of one day old, were distributed in a completely randomized design with five treatments and six replicates of 21 birds each, totaling 30 experimental units consisting of a shelter area and corrals. The levels reduced of crude protein for the initial phase were: 21.5, 21.0, 20.5, 20.0 and 19.5% and for the growth phase: 19.0, 18.5, 18.0, 17.5 and 17.0%. In all the treatments were maintained the minimum ratio between essential amino acids and lysine. The performance characteristics evaluated were: weight gain, feed intake and feed conversion. Results. There was no effect (p>0.05) of the crude protein reduction for any of the variables in all phases, except for feed conversion (p<0.05) that improved with 18.20% in the growth phase. Conclusions. Crude protein levels for chickens of the Redbro line, can be reduced to 19.5% and 18.2% for the initial and growth phases, respectively, provided that the ideal amino acid ratios are maintained with digestible lysine and meet the requirements of limiting amino acids.

Estimación del balance de metionina y lisina metabolizable en vacas de leche en pastoreo / Estimation of the balance of metabolizable lysine and methionine for grazing dairy cows

RESUMEN Objetivos. El objetivo de este trabajo fue estimar en vacas en pastoreo, tanto la reproducibilidad como la precisión del balance de metionina (Met) y lisina (Lis), aminoácidos más limitantes en la producción de leche, cuando se comparan los valores predichos versus los valores observados. Materiales y métodos. Se usaron 12 vacas durante un periodo de 20 días. Control: animales pastoreando y suplementados con alimento balanceado; Met-Lis: igual al control y el suplemento se ajustó con Lis y Met protegida. Para los valores predichos para la suplementación de Met y Lis, se tuvo en cuenta el promedio del consumo de materia seca (CMS) del hato, basado en la oferta y el consumo de pasto y los valores de proteína microbiana. Los valores observados se determinaron con base en el CMS individual usando marcadores externos e internos, además, la producción individual de proteína microbiana. Se realizó una prueba de t pareada y un modelo de predicción para determinar la eficiencia fue determinado usando el error cuadrático medio de predicción (ECMP) y el coeficiente de concordancia (CCC). Resultados. Se encontraron diferencias significativas entre la suplementación y el balance de Met y Lis, entre los valores predichos y los valores observados para el CMS del forraje, la digestibilidad de la proteína microbiana, la producción de proteína microbiana, el suministro y el balance de Lis y Met. El CCC del balance de Lis y Met fue bajo (0.10), el ECMP fue alto, excepto para el CMS, que tuvo una concordancia moderada (0.63) y un bajo ECMP(4.42). Conclusiones. Estos resultados demuestran la falta de precisión de las herramientas que se usan para balancear las raciones individuales de vacas en pastoreo, ya que subestiman la suplementación y el balance de los aminoácidos.

ABSTRACT Objective. The aim of this study was to determine in grazing cows, the reproducibility and accuracy of the balance between predicted values, when compared with the observed values for the most limiting amino acids in milk protein synthesis, the methionine (Met) and Lysine (Lys). Materials and methods. Twelve lactating cows were used for a 20-day experimental period. Control: animals grazing and supplemented with balanced food; Met-Lys: same as control and supplemented with adjusted rumen protected Met and Lys. For Met and Lys supply predicted values, it was taking in account the average of the dry matter intake (DMI) of the herd, based in offer and foraging control and values of microbial protein data. Observed values were determined based in the individual DMI intake, using external and internal markers and the individual microbial protein production. A t-paired-sample test was performed and the efficiency of the model's prediction was determined using the mean square prediction error (MSPE) and the concordance coefficient (CCC). Results. Significant differences were found between the predicted and observed values for DMI forage, digestible microbial protein and microbial protein production, supply and the balance of Lys and Met. The CCC for Lys and Met balance were low (0.10), the MSPE was high except for the total DMI with a moderate concordance (0.63) and low MSPE (4.42). Conclusions. These results indicate a lack of precision of the tools, which underestimates the supply and balance of amino acids in individual grazing cows.

Randomized controlled clinical trial of ketoanalogues supplementation in dogs with chronic kidney disease / Estudo clínico randomizado controlado da suplementação de cetoanálogos em cães com doença renal crônica

The objective was to verify the effectiveness of ketoanalogues in dogs with Chronic Kidney Disease (CKD) stage 3. Controlled randomized clinical trial was performed with twenty dogs with CKD stage 3. Animals were subjected to: Group 1 (control): conventional therapy (CT) for CKD; Group 2: CT and 60mg/kg, OA, q48h of keto-supplementa; Group 3, CT and 60mg/kg, OA, q24h of keto-supplementa; and Group 4, CT and 120mg/kg, OA, q12h of keto-supplementa. All dogs received canine renal diet. Animals were evaluated at the beginning of therapy and after 15 and 30 days. Complete blood count (CBC), serum urea, creatinine, phosphorus, calcium, potassium and sodium and urine protein/creatinine (UPC) ratio were analyzed. The use of ketoanalogues in dogs with CKD stage 3 during the period of 30 days showed no efficacy, in any of the studies dosages, to improve signs and symptoms of the disease, improve the values of CBC, reduce serum urea and creatinine, normalize electrolytes or reduce UPC. It is concluded that the use of ketoanalogues does not impact the clinical outcomes in dogs with CKD stage 3.(AU)

O objetivo foi de verificar a eficácia da suplementação com cetoanálogos em cães com Doença Renal Crônica (DRC) grau 3. Um ensaio clínico controlado e randomizado foi realizado com 20 cães com DRC grau 3. Os animais foram divididos em 4 grupos: grupo 1 (controle): terapia convencional (TC) para DRC; grupo 2: TC e 60mg/kg, VO, q48h de cetoanálogoa; grupo 3: TC e 60mg/kg, VO, q24h de cetoanálogoa; e grupo 4, TC e 120mg/kg, VO, q12h de cetoanálogoa. Todos os cães receberam ração renal para cães. Os animais foram avaliados no início da terapia e após 15 e 30 dias. Hemograma completo, ureia, creatinina, fósforo, cálcio, potássio e sódio séricos e a razão proteína creatinina (RPC) urinária foram analisados. Foi verificado que o uso dos cetoanálogos em cães com DRC grau 3 durante 30 dias não mostrou eficácia, em nenhuma das dosagens utilizadas, em melhorar os sinais clínicos e sintomatologia da doença, os valores do hemograma e ureia e creatinina séricos, normalizar eletrólitos e reduzir RPC. Concluiu-se que o uso de cetoanálogos não impacta na melhora de sintomatologia clínica em cães com CKD grau 3. Como esse parece ser o primeiro ensaio clínico sobre cetoanálogos em cães com CKD, mais estudos podem ser necessários com maior acompanhamento e maiores grupos.(AU)

Efficiency of threonine utilization in the growing pigs / Eficiencia del uso de treonina para cerdos en fase de crecimiento

Objective. This study aimed to determine the marginal efficiency of threonine utilization in growing pigs by using the nitrogen balance technique. Materials and methods. Twelve castrated pigs with an average live weight of 72±2 kg were housed in metabolic cages maintained in a temperature controlled room of 22±3°C. The treatments consisted of four diets calculated to meet 30, 45, 60 and 70% of the nutritional requirements of standardized ileal digestible threonine. The amino acids others than threonine were maintained at least 15% above threonine requirements, expressed in terms of ideal levels. The amount of feed supplied was calculate to provide 2.6 times the metabolizable energy requirements for maintenance and adjusted daily according to an expected daily gain rate of 0.8 kg. Results. Pigs consumed 1.65 g kg-1 BW0.75 nitrogen daily, on average and 61% was retained and, as a result, 39% was excreted, being 31% through the feces and the remainder (69%) in the urine. The pigs showed a linear retention of threonine in response to the increase of standardized threonine intake. For each gram of standardized threonine intake 0.37 g were lost during the metabolism. Conclusions. It is concluded that for calculating nutritional requirements of standardized threonine of pigs by the factorial method, it is possible to use the utilization marginal efficiency of 0.63.

Objetivo. El objetivo de este estudio fue determinar la eficiencia marginal del uso de treonina para cerdos en fase de crecimiento, por medio de la técnica de balance de nitrógeno. Material y métodos. Fueron utilizados doce cerdos machos castrados con peso promedio de 72±2 kg alojados en jaulas metabólicas climatizadas a una temperatura ambiente de 22±3°C. Los tratamientos consistieron en cuatro dietas calculadas para 30, 45, 60 y 70% de las necesidades nutricionales de treonina digestible estandarizada. Los demás aminoácidos se añadieron a las dietas para lograr una proporción de al menos 15% de sus necesidades expresadas con relación a la treonina. La cantidad de alimento proporcionado se calculó para suministrar 2.6 veces la energía metabolizable y ajustada todos los dias, según una tasa de ganancia media diaria de 0.8 kg. Resultados. Los cerdos consumieron 1.65 g kg-1 PV0.75 de nitrógeno por dia. En promedio 61% de nitrógeno ingerido se conservó y 39% fue excretado por el animal. El 31% se excretó en las heces y el resto (69%) en la orina. Los cerdos mostraron una retención lineal de treonina, con relación al aumento en la ingesta de treonina digestible estandarizada. Por cada gramo de treonina digestible estandarizada ingerido 0.37 g se perdieron durante el metabolismo. Conclusiones. Se concluye que, para el cálculo de las necesidades nutricionales de treonina digestible estandarizada para cerdos por método factorial, es posible utilizar la eficiencia marginal del uso de treonina a 0.63.

Suplementação de aminoácidos para redução da proteína bruta em dietas para leitões desmamados aos 21 dias de idade / Supplementation of amino acids to reduce crude protein in diets for piglets weaned at 21 days

Avaliou-se o efeito da suplementação de aminoácidos industriais para redução de proteína bruta (PB) em dietas para leitões desmamados aos 21 dias de idade sobre o desempenho e sobre as variáveis morfofisiológicas, utilizando-se 126 leitões com peso inicial de 6,05±0,35kg. Os animais foram distribuídos em delineamento experimental de blocos ao acaso, com seis tratamentos (24,0; 23,0; 22,0; 21,0; 20,0; 19,0% de PB), sete repetições e três animais por unidade experimental. Não houve efeito significativo da redução de proteína bruta da dieta sobre o ganho de peso médio diário, o consumo de ração diário e a conversão alimentar, e o pH do estômago também não sofreu influência, mas houve diminuição do pH do duodeno. Os pesos relativos do baço e do fígado, a altura de vilosidades e a profundidade de criptas no duodeno, jejuno e íleo não foram influenciados, enquanto o peso do pâncreas diminuiu com a redução da proteína bruta da dieta.

The objective of this work was to evaluate the effect of amino acid supplementation for the reduction of crude protein (CP) in diets for piglets weaned at 21 days of age on performance and morphophysiological parameters of 126 piglets with an initial weight of 6.05±0.35kg. The animals were divided into a randomized block experimental design with six treatments (24.0, 23.0, 22.0, 21.0, 20.0, 19.0% CP), seven replicates and three animals per experimental unit. It was found that there was no significant effect of reducing crude protein in the diet on the piglets' average daily weight gain, feed intake and feed conversion (FC). It was also found that the reduction of protein did not influence the pH of the stomach, however, the pH of the duodenum decreased with the reduction of CP. The relative weights of the spleen, liver and villus height and crypt depth in the duodenum, jejunum and ileum were not affected, whereas pancreatic weight decreased with the reduction of crude protein in diets.

Protein turnover, amino acid requirements and recommendations for athletes and active populations

Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers (13C-lysine, 15N-glycine, ²H5-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg-1·day-1 compared to 0.8 g·kg-1·day-1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.

Recent progress in intestinal essential amino acids metabolism and its function / 肠外与肠内营养

Dietary essential amino acids are extracted in the first-pass metabolism by the small intestine.Besides incorporation into mucosal protein,they are also the fuels for the utilization by small intestinal mucosal cells and the precursors for intestinal synthesis of new amino acids and enormous biological important molecules such as glutathione,polyamines,etc.Thus,their gut metabolism is not only crucial for the maintenance of the integrity and function of intestinal mucosa,but also important for the regulation of whole body metabolism.

Association of the Nutritional Status and Essential Amino Acids Intake in Hemodialysis Patients

The mortality and morbidity rate of hemodialysis patients (HD) remain high. Among many factors, protein and calorie malnutrition has been shown to be a major risk factor for increased mortality in the HD patients population. Malnutrition can be caused by insufficient amino acid intake, nutrient losses in dialysate, oxidant stress and muscle catabolism. In this study, we evaluated the association of markers of nutritional status and essential amino acids intake in HD patients. We investigated nutritional status of 41 HD patients (mean age: 64.2 +/- 11.5 y, men: 24, women: 27) by measuring anthropometric, biochemical parameters and food intakes by using 24 hr recall methods. Subject's total energy intake and total protein intake were 1,648.0 +/- 397.31 kcal/day, 79.2 +/- 27.2 g/day, respectively. The animal protein intake was 42.7 +/- 22.1 g/day, essential amino acids intake was 23.4 +/- 9.92 g/day, and the ratio of essential amino acids to total protein intake was 29.6 +/- 5.42%. There were significantly positive correlation between muscle mass and lean body mass with serum creatinine level (r = 0.435, p < 0.01; r = 0.435, p < 0,01). There were also significant positive correlation in muscle mass and lean body mass with pre hemodialysis blood urea nitrogen (preHD BUN)(r = 0.329, p < 0.05; r = 0.329, p < 0.05). There were no significant correlation in total energy intake and total protein intake per kg ideal body weight (IBW) to muscle mass and lean body mass. However, there were significantly positive correlation between the ratio of essential amino acids and muscle mass and lean body mass (r = 0.368, p < 0.05; r = 0.405, p < 0.01). And serum hematocrit concentration was positively correlated with the ratio of essential amino acids (r = 0.032, p < 0.05). The results of this study indicate that strong associations exist in essential amino acid intakes with malnutrition than total protein intakes in HD patient. In conclusion, specialized nutrition education should be necessary to efficiently improve the quality of protein intakes.

Expression of amino acid transport system L in the differentiation of periodontal ligament fibroblast cells / 대한치주과학회지

The periodontium is a topographically complex organ consisting of epithelial tissue, soft and mineralized tissues. Structures comprising the periodontium include the gingiva, periodontal ligament (PDL), cementum and the alveolar bone. The molecular mechanism of differentiation in PDL fibroblast cells remain unclear. Amino acid transporters play an important role in supplying nutrition to normal and cancer cells and for cell proliferation. Amino acid transport system L is a major nutrient transport system responsible for the Na+-independent transport of neutral amino acids including several essential amino acids. The system L is divided into two major subgroups, the L-type amino acid transporter 1 (LAT1) and the L-type amino acid transporter 2 (LAT2). In this study, the expression pattern of amino acid transport system L was, therefore, investigated in the differentiation of PDL fibroblast cells. To determine the expression level of amino acid transport system L participating in intracellular transport of amino acids in the differentiation of PDL fibroblast cells, it was examined by RT-PCR, observation of cell morphology, Alizaline red-S staining and uptake analysis after inducing experimental differentiation in PDL fibroblast cells isolated from mouse molar teeth. The results are as follows. 1. The LAT1 mRNA was expressed in the early stage of PDL fibroblast cell differentiation. This expression level was gradually reduced by differentiation-inducing time and it was not observed after the late stage. 2. The expression level of LAT2 mRNA was increased in time-dependent manner during differentiation induction of PDL fibroblast cells. 3. There was no changes in the expression level of 4F2hc mRNA, the cofactor of LAT1 and LAT2, during differentiation of PDL fibroblast cells. 4. The expression level of ALP mRNA was gradually increased and the expression level of Col I mRNA was decreased during differentiation of PDL fibroblast cells. 5. The L-leucine transport was reduced by time from the early stage to the late stage in PDL fibroblast cell differentiation. As the results, it is considered that among neutral amino acid transport system L in differentiation of PDL fibroblast cells, the LAT1 has a key role in cell proliferation in the early stage of cell differentiation and the LAT2 has an important role in the late stage of cell differentiation for providing cells with neutral amino acids including several essential amino acids.

Expression and functional characterization of amino acid transport system L in Saos2 human osteogenic sarcoma cells

inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). The affinity of [14C]L-leucine uptake and the inhibition profiles of [14C]L-leucine uptake by various amino acids in the Saos2 cells were comparable with those for the LAT1 expressed in Xenopus oocytes. The majority of [14C]Lleucine uptake is, therefore, mediated by LAT1 in the Saos2 cells. These results suggest that the transports of neutral amino acids including several essential amino acids into Saos2 human osteogenic sarcoma cells are for the most part mediated by LAT1. Therefore, the Saos2 human osteogenic sarcoma cells are excellent tools for examine the properties of LAT1. Moreover, the specific inhibition of LAT1 in tumor cells might be a new rationale for anti-tumor therapy.

Intestine health and metabolism of conditionally essential amino acids / 肠外与肠内营养

Integrity of gut barrier is the foundation of the multiple function of intestinal tract to keep normal.Dietary conditionally essential amino acids are major fuels for the small intestinal mucosa as well as important substrates for syntheses of intestinal proteins,nitric oxide and metabolic products,and are required for maintaining intestinal integrity.The article reviewed recent advances in dietary uses of functional amino acids(including glutamine,glutamate,and arginine) to enhance the gut health and immune status of animals.