1. Investigación

Precision nutrition in broilers requires tools capable of identifying amino acid imbalances individually or in groups, as well as knowledge on how more digestible proteins can be designed for innovative feeding programs adjusted to animals’ dynamic requirements. This work proposes two potential tools, combining traditional nutrition with biotechnological, metabolomic, computational and protein engineering knowledge, which can contribute to improving the precise amino acid nutrition of broilers in the future: (i) the use of serum uric nitrogen content as a rapid biomarker of amino acid imbalances, and (ii) the design and modeling of de novo proteins that are fully digestible and fit exactly to the animal’s requirements. Each application is illustrated with a case study. Case study 1 demonstrates that serum uric nitrogen can be a useful rapid indicator of individual or group amino acid deficiencies or imbalances when reducing dietary protein and adjusting the valine and arginine to lysine ratios in broilers. Case study 2 describes a stepwise approach to design an ideal protein, resulting in a potential amino acid sequence and structure prototype that is ideally adjusted to the requirements of the targeted animal, and is theoretically completely digestible. Both tools can open up new opportunities to form an integrated framework for precise amino acid nutrition in broilers, helping us to achieve more efficient, resilient, and sustainable production. This information can help to determine the exact ratio of amino acids that will improve the efficiency of the use of nitrogen by poultry.

The ideal protein concept refers to dietary protein with an amino acid profile that exactly meets an animal’s requirement. Low-quality protein levels in the diet have negative implications for productive and reproductive traits, and a protein oversupply is energetically costly and leads to an excessive N excretion, with potentially negative environmental impact. Urea Nitrogen (UN), which corresponds to the amount of nitrogen in the form of urea circulating in the bloodstream, is a metabolite that has been widely used to detect amino acid imbalances and deficiencies and protein requirements. This review aims to critically analyse how UN can contribute to accurately implementing the ideal protein concept in monogastric animals, particularly in pig, poultry, and rabbit nutrition (14,000 animals from 76 published trials). About 59, 37, and 4% of trials have been conducted in pigs, poultry, and rabbits, respectively. UN level was negatively correlated to main performance traits (Pearson Correlation Coefficient [PCC] of 􀀀0.98 and 􀀀0.76, for average daily gain and feed conversion ratio, respectively), and lower UN level was related to higher milk yield and concentration. High level of UN was positively correlated to N excretion (PCC = 0.99) and negatively correlated to protein retention (PCC = 􀀀0.99). Therefore, UN in blood seems to be a proper indicator of amino acid imbalance in monogastric animals. Great variability in the use of UN was observed in the literature, including uses as determination medium (blood, plasma, or serum), units, and feeding system used (ad libitum or restricted), among others. A standardization of the methods in each of the species, with the aim to harmonize comparison among works, is suggested. After review, UN measurement in plasma and, whenever possible, the utilization of the same nutritional methodology (ad libitum conditions or restriction with blood sampling after refeeding at standardised time) are recommended. More studies are necessary to know the potential of UN and other bioindicators for amino acid deficiencies evaluation to get closer to the ideal protein concept.

Quantitative and qualitative analysis of amino acids in biofluids offers relevant information in diagnosis of diseases, evaluation of nutritional state, and in elucidating metabolic influences on physiology. A simple, rapid, and robust procedure in terms of sample treatment, separation, and quantitation based on CE-LIF has been optimized for use in human plasma samples. Time required for derivatization was 15 min and analysis time was 35 min. 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was the labeling agent used for obtaining fluorescent derivatives. Electrophoretic conditions were: 175 mM borate buffer at pH 10.25 prepared with 12.5 mM -cyclodextrin. The voltage applied was +21 kV. Fourteen amino acids could be quantified: L-proline, L-phenylalanine, L-leucine, L-isoleucine, L-ornithine, D-ornithine, L-glutamine, L-alanine, L-threonine, glycine, L-serine, D-serine, taurine and L-glutamate. With this chiral CE-LIF method, L- and D-amino acids are adequately separated. The method was validated for a representative group of amino acids in human plasma: L-proline, L-isoleucine, L-ornithine, L-glutamine, L-alanine L-threonine, glycine, L-serine, D-serine, and glutamate. The method has been successfully applied to human plasma from patients with bipolar disorder, all of whom were taking lithium as a mood stabilizer. Eleven amino acids were quantified in plasma from nine patients, aged 24–55 years. The results were in accordance to published values for the bipolar patients. The method is useful particularly in studies where plasma amino acid levels can be used as biomarkers for diagnosis of diseases, evaluating the disease progression, and monitoring response to drug therapy.

This review article addresses the different chiral capillary electrophoretic methods that are being used for the study and characterization of foods and food compounds (e.g., amino acids, organic acids, sugars, pesticides). An updated overview, including works published till December 2002, on the principal applications of enantioselective procedures together with their main advantages and drawbacks in food analysis is provided. Some anticipated applications of chiral electromigration methods in food characterization are also discussed.

A 24-h fast induced different patterns of change in the amino acid concentrations of liver, muscle, plasma and blood cells. Starvation produced generalized increases in blood amino acids despite decreases in plasma, thus increasing the blood cells amino acid pool. Muscle increased amino acid levels with fasting, while the changes were much more buffered in liver. The fraction of essential amino acids carried by the blood was considerably greater than that of muscle and liver. The size of muscle pool in the whole rat was much greater than that of liver and more than two orders of magnitude higher than that of the whole blood. Fasting-induced changes agree with the known transport of amino acids from muscle and other peripheral tissues towards the liver and other splanchnic organs.

Rats chronically treated with two daily doses of tolbutamide, glibenclamide or glipentide were compared with animals treated with placebo. Plasma individual amino acids were determined at 0, 3, 7, 10, 12, 14, 17, 24, 27 and 29 days of treatment 16 hours after the administration of the drug. Rats were fasted for 48 h periods at days 10 to 12 and 27 to 29 of the experiment. Sulfonylurea treated animals show minor changes in the plasma aminogram, although glipentide and glibenclamide produced greater effects than tolbutamide. At the 3rd day after the onset of the treatment, plasma levels of glutamate+ glutamine, arginine and histidine appeared significantly reduced in glipentide and glibenclamide treated animals. When plasma samples were collected 3 h after the drug administration at the 24th day of treatment, the only observed change was a decrease in the levels of arginine in the glipentide treated animals. Fasting produced decreases in plasma levels of alanine, pro line, cysteine, tyrosine, methionine +ornithine and tryptophan, there were no changes in serine, aspartate + asparagine, threonine citruline, arginine and lysine; and glycine, glutamate+ glutamine and leucine + isoleucine show increases. These changes were rapidly compensated with refeeding, appearing a "rebound effect" in certain amino acids. Both fasting and refeeding affect very little the effect of sultonylureas on plasma amino acid levels, although for some individual amino acid they reduce or enhance the effect of the fasting. These small effect of sulfonylureas on plasma amino acid levels could be the result of the juxtaposition of different factors, including the effects of these drugs on circulating insulin levels, on protein biosynthesis and amino acids transamination and hepatic gluconeogenesis.

In this work, the development of a new chiral capillary electrophoresis-mass spectrometry (CE-MS) method to separate D- and L-amino acids is shown. On-line coupling between CE and MS is established through an electrospray-coaxial sheath flow interface. Enantiomer separation is achieved by using a cheap, nonvolatile, chiral selector as b-cyclodextrin in the background electrolyte (BGE) together with a physically coated capillary that is aimed to prevent contamination of the electrospray. The capillary coating is simple and easy to obtain as it only requires flushing of the capillary with a polymer aqueous solution for 3 min. Optimization of CE parameters (pH of BGE, type and concentration of chiral selector, and capillary inner diameter) and electrospray-MS parameters (nature and flow rate of the sheath liquid, nebulizer pressure) is carried out. Two different derivatization protocols of amino acids using dansyl chloride (DNS) and fluorescein isothiocyanate (FITC) are compared in terms of MS sensitivity and chiral resolution. Under optimum CE-MS conditions it is observed that the MS sensitivity obtained for FITC- and DNS-amino acids is similar (with limit of detection (LOD) in the mM range, corresponding to amounts injected in the fmol range) while chiral resolution is better for FITC-amino acids. The optimized method is demonstrated to provide the simultaneous analysis of 15 selected amino acids (i.e., FITC-D/L-Asp, -Glu, -Ser, -Asn, -Ala, -Pro, -Arg, and FITC-g-aminobutyric acid (GABA) in a single chiral CE-MS run, corresponding to the main amino acids that can be found in orange. Moreover, as a result of the high resolution achieved, it is possible to detect down to 2% of D-Asp in the presence of 98% of L-Asp. The good possibilities of chiral CE-MS in food analysis are corroborated through the detection of the main amino acids in a commercial orange juice (i.e., FITC-L-Asp, -Glu, -Ser, -Asn, -Pro, -Arg, and the nonchiral FITCGABA) as well as the determination of the fraudulent addition of synthetic amino acids (containing D- and L-forms) to a fresh orange juice

In this work a new method to detect the existence of chiral amino acids in orange juice is presented. The method employs â-cyclodextrins and micellar electrokinetic chromatography with laser-induced fluorescence (MEKC-LIF) to separate and detect L- and D-amino acids (L-aa and D-aa) previously derivatized with fluorescein isothiocianate (FITC). A systematic optimization of the chiral-MEKC conditions is done bringing about in less than 20 min a good separation of the main amino acids found in orange juice (i.e., Pro, Asp, Ser, Asn, Glu, Ala, Arg, and the nonchiral GABA, i.e., ç-aminobutyric acid). Using this procedure, the analysis time reproducibility for the 15 standard compounds (L-aa, D-aa, and GABA) has been determined to be better than 0.2% (n ) 5) for the same day and better than 0.7% (n ) 15) for three different days. Corrected peak area reproducibility is somewhat lower, providing values better than 3.3% (n ) 5) for the same day and 6.9% (n ) 15) for three different days. The limit of detection using this procedure was determined to be 0.86 attomoles for L-Arg. The optimized FITC derivatization method allows the easy and straightforward detection of amino acids in orange concentrates and juices (i.e., only centrifugation of diluted samples for 5 min is needed prior to their derivatization). D-Ala, D-Asp, D-Arg, and D-Glu were determined in orange juices and orange concentrates from different geographical origins using this new method. Moreover, the effect of different temperature treatments (50, 92, and 150 °C) on the content of D-aa in orange juice was evaluated.

Plasma amino acid concentrations, together with other metabolic parameters were determined in thyroidectomized rats treated with daily injections of sal ins (hypothyroid), and 250 ,ug/kg L-T4 {hypothyroid). Data were compared with sham-operated controls. TherP. is a genera! incre'!SE> in plasma amino acid concentrations in hyperthyroidism, a limited in· crease only in several amino acid r.oncentrations in hypothyroid rats as compared with controls, and a considerable difference between the plasma aminograms of both groups. Amino acid homeostasis seems to be subject to greater modification in hyperthyroidism than in hypothyroidism.