Acessibilidade / Reportar erro

Timing carbon turnover (δ13C) in weaned piglet’s brain by IRMS

Abstract

Isotope-ratio mass spectrometry (IRMS) is a potential tool that provides time-integrated estimate of assimilated and not just ingested nutrients. This feature turned possible its application to evaluate the effects of dietary nucleotides and glutamate on carbon turnover (δ13C) in the brain of weanling piglets. Eighty-seven piglets weaned at 21-day-old were used, being three piglets slaughtered at day-0 (prior to diet-switch) and, the remaining 84 randomly assigned in a complete block design with a 2 x 2 factorial arrangement of treatments (two nucleotide levels: 0% and 0.1% and, two glutamate levels: 0% and 1%). On trial days 3, 6, 9, 14, 21, 35 and 49, three piglets per treatment were also slaughtered. The samples were analyzed by IRMS and adjusted to the first-order equation by a nonlinear regression analysis using NLIN procedure of SAS, in order to obtain the exponential graphics. The carbon turnover (t95%) verified for cerebral tissue was faster (P<0.05) for diet containing glutamate in comparison to other diets, supporting the fact that glutamate contributed to develop the piglets’ brain, due to the fastest incorporation of 13C-atoms in this tissue at post-weaning phase, despite the energy deficit experienced by them.

Key words
additives; amino acids; isotopic dilution; stable isotopes; swine

INTRODUCTION

In the last decades, the stable isotopes have been used in a growing and continuous way in agricultural sciences as a promising tool to study processes related to the digestion, absorption and metabolism of nutrients in humans and animals, as well as for the purposes of meat traceability and animal’s nutrient-based dietary patterns (Gannes et al. 1998GANNES LZ, DEL-RIO CM and KOCH P. 1998. Natural abundance variations in stable isotopes and their potential uses in animal physiological ecology. Comp Biochem Physiol A Mol Integr Physiol 119: 725-737.). This technique is particularly suitable for determining the relative contribution of two isotopically distinct sources of organic matter in animal diet, providing information about the integration time of the assimilated food and not only of the ingested food (Tieszen et al. 1983TIESZEN LL, BOUTTON TW, TESDAHL KG and SLADE NA. 1983. Fractionations and turnover of stable carbon isotopes in animal tissues: Implications for δ‰13C analysis of diet. Oecologia 57: 32-37.), representing about 50% of chemical elements that constitute animals’ body (Rennie 1999RENNIE MJ. 1999. An introduction to the use of tracers in nutrition and metabolism. Proc Nutr Soc 58: 935-944.) and, highlighting its potential for nondestructive sampling, as well as alternative potential to radioactive’s isotope usage (3H- thymidine).

Body tissues reflect the isotopic signal of diets that animals consume; thus, the natural variation analysis of stable isotopes abundance represents a powerful tool for the researchers, in case of a possible replacement of diet containing animal-ingredients by plant-ingredients or vice versa. In general, fast-turnover tissues will reflect more recent diets, because they will be more metabolically active, whereas those with slower turnover rates will reflect diets from old periods and therefore, will be less metabolically active (Hobson and Clark 1992HOBSON KA and CLARK RG. 1992. Assessing avian diets using stable isotopes I: turnover of 13C in tissues. The Condor 94: 181-188.). In this context, the study of organs’ turnover is fundamental to determine how the isotopic signature of an animal’s diet is incorporated and to demonstrate how the turnover of a tissue is related to its metabolic rate (Thompson et al. 1998THOMPSON DR, BEARHOP S, SPEAKMAN JR and FURNESS RW. 1998. Feathers as a means of monitoring mercury in seabirds: insights from stable isotope analysis. Environ Pollut 101: 193-200.).

As early-weaning of piglets causes stress characterized by a decrease in feed intake followed by changes in growth rates, a fast recovery represents an essential step for proper growth of these animals. Besides that, post-weaning is a critical step for piglets, their requirement for nucleotides is higher; however, its percursors’ availability (energy and glutamine) are at low levels. Previous studies have assessed a potential role for dietary glutamic acid and nucleotides as performance-enhancing additives (Lackeyram et al. 2001LACKEYRAM D, YUE X and FAN MZ. 2001. Effects of dietary supplementation of crystalline L-glutamine on the gastrointestinal tract and whole body growth in early-weaned piglets fed corn and soybean meal-based diets. J Anim Sci 79(Suppl.1): 322., Rezaei et al. 2013REZAEI R, KNABE DA, TEKWE CD, DAHANAYAKA S, FICKEN MD, FIELDER SE, EIDE SJ, LOVERING SL and WU G. 2013. Dietary supplementation with monosodium glutamate is safe and improves growth performance in postweaning pigs. Amino Acids 44: 911-923., Weaver and Kim 2014WEAVER AC and KIM SW. 2014. Supplemental nucleotides high in inosine 5′-monophosphate to improve the growth and health of nursery pigs. J Anim Sci 92: 645-651.). Therefore, the dietary supplementation of glutamate and nucleotides could present an additive-enhancing action and/or a health improvement for weanling piglets; although, information regarding these additives with young animals is scarce, as well as, the studies carried out with pigs at this phase have been hard to unveil due to technical limitations.

Glutamate is a multifunctional amino acid that acts as an essential substrate of intermediate metabolism, being important especially for skeletal muscle, brain, kidneys and liver (serves as a major energy substrate) (Novak et al. 1994NOVAK DA, CARVER JD and BARNESS LA. 1994. Dietary nucleotides affect hepatic growth and composition in the weanling mouse. J Parenter Enteral Nutr 18: 62-66., Yamamoto et al. 1997YAMAMOTO S, WANG MF, ADJEI AA and AMEHO CK. 1997. Role of nucleosides and nucleotides in immune system, gut reparation after injury and brain function. Nutrition 13: 372-374., Newsholme et al. 2003NEWSHOLME P, PROCOPIO J, LIMA MMR, PITHON-CURI TC and CURI R. 2003. Glutamine and glutamate - their central role in cell metabolism and function. Cell Biochem Funct 21: 1-9.). Besides that, it activates taste receptors in stomach (Brosnan 2000BROSNAN JT. 2000. Glutamate at the interface between amino acid and carbohydrate metabolism. J Nutr 130: 988-990.) and, it is classified as an excitatory neurotransmitter (Liu et al. 2002LIU T, PENG J, XIONG Y, ZHOU S and CHENG X. 2002. Effects of dietary glutamine and glutamate supplementation on small intestinal structure, active absorption and DNA, RNA concentrations in skeletal muscle tissue of weaned piglets during d 28 to 42 of age. Asian-Australian J Anim Sci 15: 238-242.). On the other hand, nucleotides are important in providing nitrogen bases and nucleosides for tissues deficient on nucleotide synthesis. They have also shown to benefit mitotic rates (Sauer et al. 2011SAUER N, MOSENTHIN R and BAUER E. 2011. The role of dietary nucleotides in single-stomached animals. Nutr Res Rev 24: 46-59.) and the digestive tract development (Giacometti 1979GIACOMETTI T. 1979. Glutamic acid, advances in biochemistry and physiology, 1st ed., New York: Raven Press, 400 p., Domeneghini et al. 2004DOMENEGHINI C, DI GIANCAMILLO A, SAVOINI G, PARATTE R, BONTEMPO V and DELL’ORTO V. 2004. Structural patterns of swine ileal mucosa following L-glutamine and nucleotide administration during the weaning period. A histochemical and histometrical study. Histol Histopathol 19: 49-58., Amorim et al. 2017AMORIM AB, BERTO DA, SALEH MAD, MIASSI GM and DUCATTI C. 2017. Dietary glutamine, glutamic acid and nucleotides increase the carbon turnover (δ13C) on the intestinal mucosa of weaned piglets. Animal 11: 1472-1481.). Considering the aforementioned, this study aimed at evaluating the influence of dietary glutamic acid and nucleotides on carbon turnover (δ13C) in piglets’ brain using isotope-ratio mass spectrometry (IRMS) as a tool for isotopic characterization of diets and cerebral tissue.

MATERIALS AND METHODS

The experiment was carried out at swine production area at Faculty of Veterinary Medicine and Animal Science from São Paulo State University (UNESP), Botucatu Campus with 87 piglets of a crossbred commercial lineage (Landrace x Large White) weaned at 21 days of age and an average weight of 6.30 ± 0.13 kg. All experimental procedures were previously approved by the Animal Ethics Committee of São Paulo State University (UNESP) under protocol number 141/2014 and, in accordance with directive 2010/63/EU.

The 84 animals (one piglet per pen with different weight categories: light, medium and heavy) were housed in a nursery facility with a ceiling height of 3.5 m and lateral curtains and, suspended in metal pens with an area of 1.0 m x 1.75 m, equipped with one feeder (with three compartments for feeding), one nipple-type drinker and, one heater. The pens had a partially slatted plastic flooring, and a compact concrete floor under the heater. A water layer under the pens was replaced once or twice a week depending on volume of waste produced. The internal temperature of nursery facility was controlled by adjustment of lateral curtains and management of heaters.

Piglets were fed ad libitum within a phase feeding program to attend its nutritional requirements, in accordance with Rostagno et al. (2011)ROSTAGNO HS, ALBINO LFT, DONZELE JL, GOMES PC, OLIVEIRA RF, LOPES DC, FERREIRA AS, BARRETO SLT and EUCLIDES RF. 2011. Brazilian tables for poultry and swine: composition of feedstuffs and nutritional requirements, 3rd ed., Viçosa: Universidade Federal de Viçosa, 252 p., as follows: pre-starter I (21 to 35 days), pre-starter II (36 to 50 days) and starter diets (51 to 70 days). The evaluated treatments were additive-free diet: control (C); diet containing 0.1% nucleotides (Nu) (PubChem CID: 21712, 20819); diet containing 1% glutamic acid (GA) (PubChem CID: 33032) and, diet containing the mixture of 0.1% Nu and 1% GA (Nu+GA) presented in Table I.

TABLE I
Percentual composition and calculated nutritional values of experimental diets.

The source of nucleotides (98% of chemical purity) was constituted by disodium guanylate and disodium inosinate. The main energy source of these diets was rice grits, a raw ingredient coming from the C3-photosynthetic cycle, which showed a 13C-isotopic signal distinct from diets provided to sows, because gestation and lactation diets primarily contained corn (a C4-photosynthetic cycle), as energy source.

At baseline (day-0), three piglets were slaughtered, in order to express the isotopic composition of the tissue, which was a function of diets provided to sows in farrowing phase. The remaining piglets (84) were randomly allotted to a block design with 2 x 2 factorial arrangement of treatments (two levels of nucleotides: 0% and 0.1% and, two levels of glutamic acid: 0% and 1%) and at days 3, 6, 9, 14, 21, 35 and, 49 post-weaning, three piglets per treatment (one piglet from each weight category) were slaughtered after electronarcosis. Then, a 4-cm2 area from brain was sampled, washed with de-ionized water and packed into plastic bags, labeled and immediately frozen (-18°C) for further isotopic analyses.

In order to perform the isotopic analyses, the samples were previously defrosted and dried in a circulating-air oven (model MA-035/5, Marconi, Piracicaba, Brazil) at 50°C for 48-hour. As the lipid fraction might cause isotopic fractionation up to 5‰ of 13C values (Piasentier et al. 2003PIASENTIER E, VALUSSO R, CAMIN F and VERSINI G. 2003. Stable isotope ratio analysis for authentication of lamb meat. Meat Sci 64: 239-247.), the samples were degreased with ethyl ether (chemically pure) in a Soxhlet apparatus (model TE-044, Tecnal, Piracicaba, Brazil) at 65°C for 4-hour and submitted again to drying at 50°C for 30-min to removal of ethyl ether residual. Then, samples were cryogenic milled (model 2010, SPEX SamplePrep Geno/Grinder, Metuchen, USA) as follows: diets and ingredients: 8 cycles of 6-min at 1,000 rpm, brain: 1 cycle of 6-min at 990 rpm under -190°C, in order to obtain homogeneous material (particle size <60 μm) and to avoid repeatability problems at analyses’ step.

The samples (50 to 70 μg) were weighted in an analytical micro balance (model XP26, Mettler Toledo, Columbus, USA) into tin capsules, which were then introduced by means of an automatic sampler (model AS-3000, Thermo Fisher Scientific, Waltham, USA) in an elemental analyzer (model Flash CHNS/O 2000, Thermo Fisher Scientific, Waltham, USA) where, under the presence of oxygen, each sample was quantitatively combusted to obtain CO2 following the isotopic gas combinations and their respective m/z: 12C + 16O + 16O = 44; 13C + 16O + 16O = 45, and 13C + 16O + 16O = 46. The generated gases were separated on a gas chromatographic column (PN 26008205, Thermo Scientific, Germany) maintained at 45°C and analyzed on an isotope ratio mass spectrometer (IRMS - Delta V Advantage, Thermo Fisher Scientific, Waltham, USA) interconnected to the software Isodat 3.0 (Isomass Scientific, Calgary, Canada) at the Center of Environmental Stable Isotopes, Biosciences Institute of UNESP, Botucatu Campus.

The mass spectrometer calibration was performed with CO2 6.0 (99.99% of chemical purity) from a cylinder, which was previously calibrated against the official reference material IVA-33802174 (certificate number: 147273, Meerbusch, Germany), consisting of a certified urea, which provided a value of δ13CV-PDB = -39.73‰ ± 0.2‰. This certified urea was used for a 6-month period to ensure the spectrometer calibration. The 13C/12C standard isotope ratio was expressed as δ (delta) ‰ (per thousand) relative to the PDB (Pee Dee Belemnite), the primary standard. At the beginning of each run, two pulses of CO2 reference gas were admitted in the system for about 5 s. The constant flow rate during this period has given peaks a flat-top appearance. A level of CO2 corresponding to 5 V at m/z 44 was used to calibrate the system.

For internal quality control of analyses, during each cycle of readings (at the beginning and the end of each 45-sample sequential analyses), an internal standard was used to ensure the accuracy of quantifications. The internal standard was a coal from eucalyptus-plant, calibrated and analyzed against the working standard (IVA). The averaged δ13C-signal obtained after triplicate analyses of the coal was -28.00‰ ± 0.2‰. Samples were analyzed in duplicate and, standard analyzed in four replicates and then, the values averaged. The precision of method was set at 12‰ that refers to the amplitude of isotopic signals between C3- and C4-plants. The certainty of method was set as 0.2‰ of standard deviation.

The spectrometer readings were expressed as δ13CV-PDB (‰) notation in relation to the PDB international standard with analysis deviation at the order of 0.2‰, calculated by the equation:

δ13C(sample, standard) = {[(13C/12C)sample - (13C/12C)standard] / (13C/12C)standard} x 103 that can be summarized with the following equation: δ13C(sample, standard) = [(Rsample / Rstandard) – 1] x 103, where: δ13C = the enrichment of the isotopic ratio 13C/12C of the sample to the standard (‰); R = the ratio of the heavier (13C) to the lighter (12C) stable isotopes (dimensionless).

To evaluate the carbon substitution rate of samples, the following exponential function over time was employed (Ducatti et al. 2002DUCATTI C, CARRIJO AS, PEZZATO AC and MANCERA PFA. 2002. Theoretical and experimental model for carbon-13 turnover in mammalian and avian tissues. Sci Agric 59: 29-33.): δ13C(t) = δ13C(f) + (δ13C(i) - δ13C(f)) e-kt, where: δ13C(t) = isotopic enrichment of tissue at any time (t); δ13C(f) = isotopic enrichment of tissue at the equilibrium or final condition; δ13C(i) = isotopic enrichment of tissue at the beginning; k = turnover constant (days), and t = time (days) since diet switch (C4- to C3-based diet).

The carbon half-life (T50%) of samples at t = T necessary for initial atoms substitution by the final atoms was determined by the equation (Ducatti et al. 2002DUCATTI C, CARRIJO AS, PEZZATO AC and MANCERA PFA. 2002. Theoretical and experimental model for carbon-13 turnover in mammalian and avian tissues. Sci Agric 59: 29-33.): tn% = (-1 / k) ln (1 - F), where: t = time of initial atoms substitution by the final substitution (days), ln = natural logarithm, F = value of atom substitution, which can vary among 0.0 to 0.95, and k = turnover constant (days).

It means that when F = 0.50, there is 50% of carbon substitution, thus the half-life value (T50%) is achieved, which gives the estimated organ turnover. The half-life equation allows us to infer that tissues, which provide lower half-life values will present a higher metabolization or incorporation of stable isotopes; the opposite being true. In contrast, when F = 0.95, there is 95% of carbon substitution (t95%), that is, the isotopic turnover was stabilized, so the tissue has reached its isotopic-plateau level (Ducatti et al. 2016DUCATTI C, SARTORI MMP, DENADAI JC, COSTA VE, PELICIA VC and MACARI M. 2016. Modelling and determination of metabolic pools by stable carbon isotopes in the avian duodenal mucosa and albumen. J Anim Physiol Anim Nutr 100: 77-84.). The calculation of the isotopic substitution plateau reflects the reliability in half-life values, in order to assess if experimental period was enough for 13C-substitution in a particular tissue. By this way, researchers can use the half-life parameter to compare their treatments (Table II).

TABLE II
Half-life (T50%) and 13C total substitution (t95%) values of piglets’ brain according to experimental diets.

The carbon isotopic enrichment data (δ13C, ‰) analyzed by IRMS were compiled versus each slaughter day throughout 49 days. Subsequently, each set of data (slaughter day versus brain-δ13C from each piglet) were adjusted to the first-order equation by a nonlinear regression analysis using the NLIN of SAS (SAS Institute, Cary, NC, USA) in order to establish the δ13C over time. The exponential graphics were plotted by software OriginPro 8.07 (OriginLab Corporation, Northampton, MA, USA) to further calculations and comparison between half-life values (T50%),) and t95%), of treatments that were subjected to analysis of variance using the GLM(general linear model) procedure of the SAS software, v.9.2 (SAS Institute Inc., Cary, NC, USA), and the means compared according to each weight category of piglets by Tukey’s test at 5% of significance.

RESULTS

The diet provided to lactating sows, as well as, to suckling piglets reflected the isotopic signature of C4),-plant, presenting a mean value of δ13C = -16.15‰, due to the main ingredient of this diet was corn, a C4-plant, which when analyzed, presented a mean value of δ13C = -17.40‰. The isotopic discrimination between cereals were observed in this study, due to alteration over time of diet’s isotopic signature from corn to rice, which has given diet mean values of δ13CV-PDB, as follow: -27.59‰, -27.86‰, -27.40‰ and, -27.57‰ in C-diets; Nu-diets; GA-diets and, Nu+GA-diets, respectively.

The brain of piglets fed Nu-diet presented the slowest carbon turnover value (P<0.05) (Table II), due to 81% of carbon-13 substitution within 49 days, resulting on higher half-life (T50%),) and 95% of 13C-substitution (t95%),) values. Besides that, cerebral tissue of piglets fed C-diet needed almost 7-day less than those fed the Nu-diet to incorporate 83% of carbon-13 atoms during the same period (2% more carbon-13 atoms substituted than Nu-diet), which has favored the reduction (P<0.05) of its t95%-value.

However, piglets fed Nu+GA-diet had 84% of carbon-13 atoms incorporation in their brain, providing T50%-value of 7.44-h faster than piglets fed C-diet and 1-d and 22-h faster than animals fed Nu-diet. In addition, the Nu+GA-diet determined the second lower t95%-value (P<0.05) compared to other diets, which probably occurred due to the presence of glutamate, because piglets fed GA-diet presented the fastest (P<0.05) brain turnover (t95%) in relation to piglets fed other diets, so that carbon-13 atoms incorporation during experimental period was 84%, culminating in T50%- and t95%-values, respectively, 14-h and 2.5-d faster than those verified for C-diet.

Regarding isotopic composition of brain, Figure 1 shows that trial duration was not sufficient for cerebral tissue to reach the isotopic plateau, because the obtained t95%-values were higher than 49 days (Table II). Despite this, the experimental period was sufficient for the isotopic dilution’s occurrence in this organ, because δ13CV-PDB values of piglet’s brain on day-0 reflected isotopic signals averaged -15.83‰, characteristic signal of corn (C4-plant: -16.15‰) and, due to δ13CV-PDB value verified in this tissue at the end of experiment (day-49) on average was -23.86‰, characteristic signal of rice (C3-plant: -30.14‰) provided to piglets of 21- to 70-day-old.

Figure 1
Non-linear adjustment of exponential curves (δ‰13CV-PDB) of piglets’ brain (a): control diet, (b): 0.1% nucleotides, (c): 1% glutamic acid, and (d): 0.1% nucleotides + 1% glutamic acid. Values expressed as: mean (n = 3) ± standard error.

DISCUSSION

The results of experimental diets (Table I) demonstrated that isotopic signals were close to δ13C = -27‰, as expected, because they reflected the isotopic signature of rice, indicating the isotopic alteration as a function of diets provided to piglets (Smith and Epstein 1971SMITH BN and EPSTEIN S. 1971. Two categories of 13C/12C ratios for higher plants. Plant Physiol 47: 380-384.), as well as the isotopic discrimination between cereals (corn versus rice) (Park and Epstein 1961PARK R and EPSTEIN S. 1961. Metabolic fractionation of 13C and 12C in plants. Plant Physiol 36: 133-138.), allowing us to state that principles of isotopic dilution (Hobson and Clark 1992HOBSON KA and CLARK RG. 1992. Assessing avian diets using stable isotopes I: turnover of 13C in tissues. The Condor 94: 181-188., Jones et al. 1979JONES RJ, LUDLOW MM, TROUGHTON JH and BLUNT CG. 1979. Estimation of the proportion of C3 and C4 plants species in the diet of animals from the ratio of natural 12C and 13C isotopes in the faeces. J Agric Sci 92: 91-100., Tieszen et al. 1983TIESZEN LL, BOUTTON TW, TESDAHL KG and SLADE NA. 1983. Fractionations and turnover of stable carbon isotopes in animal tissues: Implications for δ‰13C analysis of diet. Oecologia 57: 32-37.) were achieved in this experiment and also that isotopic composition of diets can be estimated by carbon-13 isotopic value of piglet’s brain. The obtained results corroborate those reported by DeNiro and Epstein (1978)DENIRO MJ and EPSTEIN S. 1978. Influence of diet on the distribution of carbon isotopes in animals. Geochim Cosmichim Acta 42: 495-506. that verified the organic matter analysis of diet results in a more reliable measurement of isotopic data (δ13C, ‰) in relation to other biochemical fractions, as the lipid, carbohydrate and the protein.

The role of glutamate in cerebral metabolism is very important because it is the major excitatory neurotransmitter in the central nervous system of mammals and it is the precursor of γ-aminobutyric acid (GABA), a predominantly inhibitory neurotransmitter (Mangia et al. 2012MANGIA S, GIOVE F and DINUZZO M. 2012. Metabolic pathways and activity-dependent modulation of glutamate concentration in the human brain. Neurochem Res 37: 2554-2561.). Also, it is the only amino acid oxidized by the brain, because the enzyme glutamate-deaminase acts by catalyzing the reverse reaction (the production of glutamate from ammonia and α-ketoglutarate) that in turn, accelerates the carbohydrate metabolism (Weil-Malherbe 1936WEIL-MALHERBE H. 1936. Studies in brain metabolism. I. The metabolism of glutamic acid in brain. Biochem J 30: 665-676.), demonstrating the anaplerotic action of glutamate in Krebs’ cycle.

In addition, the study of Nachmansohn et al. (1943)NACHMANSOHN D, JOHN HM and WAELSCH H. 1943. Effect of glutamic acid on the formation of acetylcholine. J Biol Chem 150: 485-486. reported that dietary L-glutamate supplementation increased 4 to 5 times acetylcholine synthesis, demonstrating its key role in brain function. Other study verified the role of glutamic acid on increased learning of rats fed diets containing L-glutamate in relation to those fed a control diet, as well as the increased percentage of rats able to overcome obstacles to access the diet (Zimmerman and Ross 1944ZIMMERMAN FT and ROSS S. 1944. Effect of glutamic acid and other amino acids on maze learning in the white rat. Arch Neur Psych 51: 446-451.), and increased intelligence score of children with intellectual disability who consumed 12 g day-1 of L-glutamate for 6 months that have showed significant improvements in verbal and motor performance tests, as well as changes in traits of their personality (Zimmerman et al. 1947ZIMMERMAN FT, BURGEMEISTER BB and PUTNAM TJ. 1947. A group study of the effect of glutamic acid upon mental functioning in children and adolescents. Psychosom Med 9: 175-183.).

Gibbs et al. (2007)GIBBS ME, LLOYD HGE, SANTA T and HERTZ L. 2007. Glycogen is a preferred glutamate precursor during learning in 1-day-old chick: biochemical and behavioral evidence. J Neurosci Res 85: 3326-3333. demonstrated that glycogen is the preferred precursor of glutamate by nervous cells, the astrocytes (through α-ketoglutarate transamination) and neurons (through glutamine hydrolysis) of 1-day-old chicks and, that glutamate has been involved since the early days of life in the learning process of birds. In addition, a research with early-weaned piglets supplemented with 6.51% L-glutamate of analytical purity during 12 days reported an improvement in total growth of small intestine, as well as in duodenal mucosa, preventing atrophy of duodenum villi that normally occurs after weaning compared to piglets fed a control diet or a diet supplemented with polyamines (Ewtushik et al. 2000EWTUSHIK AL, BERTOLO RFP and BALL RO. 2000. Intestinal development of early-weaned piglets receiving diets supplemented with selected amino acids or polyamines. Can J Anim Sci 80: 653-662.).

Moreover, considering the affinity of glutamate for brain tissue and the fact that several studies have demonstrated its importance, it is possible that in the present study, its supplementation may have contributed to the brain growth (protein synthesis) (Platt 2007PLATT SR. 2007. The role of glutamate in central nervous system health and disease – a review. Vet J 173: 278-286.) due to the fastest turnover (t95%) observed, because glutamate is the only amino acid capable of serving as a source of oxoglutarate to maintain brain activity regardless of glucose levels (Weil-Malherbe 1936WEIL-MALHERBE H. 1936. Studies in brain metabolism. I. The metabolism of glutamic acid in brain. Biochem J 30: 665-676., Woodman and McIlwain 1961WOODMAN RJ and MCILWAIN H. 1961. Glutamic acid, other amino acids and related compounds as substrates for cerebral tissues: their effects on tissue phosphates. Biochem J 81: 83-93.), it has post-synaptic action (Tapiero et al. 2002TAPIERO H, MATHÉ G, COUVREUR P and TEW KD. 2002. II. Glutamine and glutamate. Biomed Pharmacother 56: 446-457.) and it is oxidized by the brain to release α-ketoglutarate (for glucose synthesis through Krebs’ cycle) and ammonia (through transdeamination) (Nelson and Cox 2012NELSON DL and COX MM. 2012. Lehninger Biochemistry Principles, 6th ed., London: Macmillan Publishers Ltd, 1340 p.).

In conclusion, the supplementation of 1% glutamic acid was efficient to develop the brain of piglets, despite the energy deficit experienced by them at post-weaning period. However, further research is needed to evaluate the economic feasibility of its inclusion in commercial diets and its most adequate supply period. To date, IRMS for timing carbon turnover (δ13C) is an important methodology in animal nutrition’ studies (e.g. obtaining biomolecules as biomarkers or as substitute for radioisotopes’ usage).

ACKNOWLEGMENTS

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) under grant numbers: 2014/13169-6 and 2013/25616-4.

REFERENCES

  • AMORIM AB, BERTO DA, SALEH MAD, MIASSI GM and DUCATTI C. 2017. Dietary glutamine, glutamic acid and nucleotides increase the carbon turnover (δ13C) on the intestinal mucosa of weaned piglets. Animal 11: 1472-1481.
  • BROSNAN JT. 2000. Glutamate at the interface between amino acid and carbohydrate metabolism. J Nutr 130: 988-990.
  • DENIRO MJ and EPSTEIN S. 1978. Influence of diet on the distribution of carbon isotopes in animals. Geochim Cosmichim Acta 42: 495-506.
  • DOMENEGHINI C, DI GIANCAMILLO A, SAVOINI G, PARATTE R, BONTEMPO V and DELL’ORTO V. 2004. Structural patterns of swine ileal mucosa following L-glutamine and nucleotide administration during the weaning period. A histochemical and histometrical study. Histol Histopathol 19: 49-58.
  • DUCATTI C, CARRIJO AS, PEZZATO AC and MANCERA PFA. 2002. Theoretical and experimental model for carbon-13 turnover in mammalian and avian tissues. Sci Agric 59: 29-33.
  • DUCATTI C, SARTORI MMP, DENADAI JC, COSTA VE, PELICIA VC and MACARI M. 2016. Modelling and determination of metabolic pools by stable carbon isotopes in the avian duodenal mucosa and albumen. J Anim Physiol Anim Nutr 100: 77-84.
  • EWTUSHIK AL, BERTOLO RFP and BALL RO. 2000. Intestinal development of early-weaned piglets receiving diets supplemented with selected amino acids or polyamines. Can J Anim Sci 80: 653-662.
  • GANNES LZ, DEL-RIO CM and KOCH P. 1998. Natural abundance variations in stable isotopes and their potential uses in animal physiological ecology. Comp Biochem Physiol A Mol Integr Physiol 119: 725-737.
  • GIACOMETTI T. 1979. Glutamic acid, advances in biochemistry and physiology, 1st ed., New York: Raven Press, 400 p.
  • GIBBS ME, LLOYD HGE, SANTA T and HERTZ L. 2007. Glycogen is a preferred glutamate precursor during learning in 1-day-old chick: biochemical and behavioral evidence. J Neurosci Res 85: 3326-3333.
  • HOBSON KA and CLARK RG. 1992. Assessing avian diets using stable isotopes I: turnover of 13C in tissues. The Condor 94: 181-188.
  • JONES RJ, LUDLOW MM, TROUGHTON JH and BLUNT CG. 1979. Estimation of the proportion of C3 and C4 plants species in the diet of animals from the ratio of natural 12C and 13C isotopes in the faeces. J Agric Sci 92: 91-100.
  • LACKEYRAM D, YUE X and FAN MZ. 2001. Effects of dietary supplementation of crystalline L-glutamine on the gastrointestinal tract and whole body growth in early-weaned piglets fed corn and soybean meal-based diets. J Anim Sci 79(Suppl.1): 322.
  • LIU T, PENG J, XIONG Y, ZHOU S and CHENG X. 2002. Effects of dietary glutamine and glutamate supplementation on small intestinal structure, active absorption and DNA, RNA concentrations in skeletal muscle tissue of weaned piglets during d 28 to 42 of age. Asian-Australian J Anim Sci 15: 238-242.
  • MANGIA S, GIOVE F and DINUZZO M. 2012. Metabolic pathways and activity-dependent modulation of glutamate concentration in the human brain. Neurochem Res 37: 2554-2561.
  • NACHMANSOHN D, JOHN HM and WAELSCH H. 1943. Effect of glutamic acid on the formation of acetylcholine. J Biol Chem 150: 485-486.
  • NELSON DL and COX MM. 2012. Lehninger Biochemistry Principles, 6th ed., London: Macmillan Publishers Ltd, 1340 p.
  • NEWSHOLME P, PROCOPIO J, LIMA MMR, PITHON-CURI TC and CURI R. 2003. Glutamine and glutamate - their central role in cell metabolism and function. Cell Biochem Funct 21: 1-9.
  • NOVAK DA, CARVER JD and BARNESS LA. 1994. Dietary nucleotides affect hepatic growth and composition in the weanling mouse. J Parenter Enteral Nutr 18: 62-66.
  • PARK R and EPSTEIN S. 1961. Metabolic fractionation of 13C and 12C in plants. Plant Physiol 36: 133-138.
  • PIASENTIER E, VALUSSO R, CAMIN F and VERSINI G. 2003. Stable isotope ratio analysis for authentication of lamb meat. Meat Sci 64: 239-247.
  • PLATT SR. 2007. The role of glutamate in central nervous system health and disease – a review. Vet J 173: 278-286.
  • RENNIE MJ. 1999. An introduction to the use of tracers in nutrition and metabolism. Proc Nutr Soc 58: 935-944.
  • REZAEI R, KNABE DA, TEKWE CD, DAHANAYAKA S, FICKEN MD, FIELDER SE, EIDE SJ, LOVERING SL and WU G. 2013. Dietary supplementation with monosodium glutamate is safe and improves growth performance in postweaning pigs. Amino Acids 44: 911-923.
  • ROSTAGNO HS, ALBINO LFT, DONZELE JL, GOMES PC, OLIVEIRA RF, LOPES DC, FERREIRA AS, BARRETO SLT and EUCLIDES RF. 2011. Brazilian tables for poultry and swine: composition of feedstuffs and nutritional requirements, 3rd ed., Viçosa: Universidade Federal de Viçosa, 252 p.
  • SAUER N, MOSENTHIN R and BAUER E. 2011. The role of dietary nucleotides in single-stomached animals. Nutr Res Rev 24: 46-59.
  • SMITH BN and EPSTEIN S. 1971. Two categories of 13C/12C ratios for higher plants. Plant Physiol 47: 380-384.
  • TAPIERO H, MATHÉ G, COUVREUR P and TEW KD. 2002. II. Glutamine and glutamate. Biomed Pharmacother 56: 446-457.
  • THOMPSON DR, BEARHOP S, SPEAKMAN JR and FURNESS RW. 1998. Feathers as a means of monitoring mercury in seabirds: insights from stable isotope analysis. Environ Pollut 101: 193-200.
  • TIESZEN LL, BOUTTON TW, TESDAHL KG and SLADE NA. 1983. Fractionations and turnover of stable carbon isotopes in animal tissues: Implications for δ‰13C analysis of diet. Oecologia 57: 32-37.
  • WEAVER AC and KIM SW. 2014. Supplemental nucleotides high in inosine 5′-monophosphate to improve the growth and health of nursery pigs. J Anim Sci 92: 645-651.
  • WEIL-MALHERBE H. 1936. Studies in brain metabolism. I. The metabolism of glutamic acid in brain. Biochem J 30: 665-676.
  • WOODMAN RJ and MCILWAIN H. 1961. Glutamic acid, other amino acids and related compounds as substrates for cerebral tissues: their effects on tissue phosphates. Biochem J 81: 83-93.
  • YAMAMOTO S, WANG MF, ADJEI AA and AMEHO CK. 1997. Role of nucleosides and nucleotides in immune system, gut reparation after injury and brain function. Nutrition 13: 372-374.
  • ZIMMERMAN FT, BURGEMEISTER BB and PUTNAM TJ. 1947. A group study of the effect of glutamic acid upon mental functioning in children and adolescents. Psychosom Med 9: 175-183.
  • ZIMMERMAN FT and ROSS S. 1944. Effect of glutamic acid and other amino acids on maze learning in the white rat. Arch Neur Psych 51: 446-451.

Publication Dates

  • Publication in this collection
    26 July 2018
  • Date of issue
    Aug 2018

History

  • Received
    27 Oct 2017
  • Accepted
    3 Jan 2018
Academia Brasileira de Ciências Rua Anfilófio de Carvalho, 29, 3º andar, 20030-060 Rio de Janeiro RJ Brasil, Tel: +55 21 3907-8100 - Rio de Janeiro - RJ - Brazil
E-mail: aabc@abc.org.br