In view of the importance of alanine as a gluconeogenic precursor in ruminants, the objective of the present study was to assess the effectiveness of three techniques in estimating the metabolic parameters surrounding alanine in wethers fed a maintenance diet of alfalfa hay. A preliminary experiment utilized a blood flow technique to study the net production and/or utilization of both alanine and glucose by the portal drained viscera. Such a method involved evaluating the arterio-venous concentration differences of alanine and glucose, in conjunction with determining the rate of portal vein blood flow. Radioactively labelled ¹⁴C - alanine was administered as a single injection in the second series of experiments to estimate the metabolic parameters of alanine as well as its contribution to glucose synthesis. The L-U-¹⁴C-alanine was given intravenously through previously implanted jugular catheters and the fall in the specific activity of plasma alanine with time was determined. The line of best fit for the decay curve of the specific activity of plasma alanine was constructed by means of a computer using a multi-term exponential function which enables the estimation of such parameters as the pool size, space, total entry rate, irreversible loss and recycling of alanine. The per cent conversion of alanine to glucose was determined by the corresponding peak of glucose specific activity following the single injection of ¹⁴C - alanine. The turnover of alanine was also studied using a continuous infusion of L-U-¹⁴C-alanine without a priming injection. The specific activity of plasma alanine reached a plateau five hours after the beginning of the infusion. It was from these plateau levels that the rate of irreversible loss of alanine as well as its percent conversion to glucose was estimated. The results indicated that the single injection technique was able to partition the total entry rate of alanine into irreversible loss and recycling and thus prove more informative than a continuous infusion method. The present study also suggested that under certain physiological stress conditions in ruminants, where recycling becomes prominent, a continuous infusion approach may overestimate the actual rate of irreversible loss of alanine. / Land and Food Systems, Faculty of / Graduate

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<p>Muscle fatigue has always been of vital importance in most sports. A few possible factors have been reported to be the cause of muscular fatigue during high intensity exercise; depletion of glycogen, oxidative stress, disruption of contractile mechanisms and accumulation of metabolites. One of the theories of the cause of muscular fatigue, both in endurance and intermittent sports, is decreased pH levels due to increased concentration of H+ ions dissociated from lactic acid in muscle. Carnosine, a fairly unnoticed ergogenic aid, taken in the form of β-alanine has shown to potentially delay the onset of fatigue. Supplementation of β-alanine, would increase carnosine levels in muscle and may counteract the decrease in pH since carnosine functions as a H+ buffer. The purpose of the present study was to examine the effects of 8 weeks supplementation of β-alanine in distance runners and Swedish division four soccer players on aerobic capacity, intermittent recovery and muscular fatigue. The runners (n = 15) were tested in lactate profiling tests and the soccer players (n = 22) were tested in the Yo-Yo intermittent endurance test pre and post the 8-week test-period. The yo-yo test did not result in significant difference between the soccer players’ β-group and control-group (p = 0,29). Neither did the lactate test result in significant differences between the distance runners’ β-group and control-group in any of the five variables measured. However, a trend in difference was seen between groups in both velocity at lactate threshold (VLT) (p = 0,11) and recovery blood lactate (RBL) (p = 0,14) where the β-group had increased slightly from 16,8 ± 1,6 km/h to 17,0 ± 1,2 km/h in VLT and decreased from 4,5 ± 1,6 mmol∙L-1 to 3,1 ± 1,0 mmol∙L-1 in RBL. The results suggested that β-alanine may delay the onset of fatigue and improve performance in endurance sports such as running by increasing the removal of lactate acid from muscle.</p>

Early research with beta-alanine (beta-ALA) supplementation has shown increases in muscle carnosine as well as improvements in body composition, exercise performance and blood lactate levels. Creatine monohydrate supplementation has been extensively researched for its effects on anaerobic exercise performance. Recently, a new line of studies have examined the combined effects beta-ALA and creatine supplementation on anaerobic exercise performance and lactate threshold. The purpose of the present study is to examine the acute and chronic effects of beta-ALA supplementation with and without creatine monohydrate on body composition, aerobic and anaerobic exercise performance, and muscle carnosine and phosphagen levels in college-aged recreationally active females. Thirty-two females were randomized in a double-blind placebo controlled manner into one of four supplementation groups including beta-ALA only, creatine only, beta-ALA and creatine combined and placebo. Participants supplemented for four weeks and reported for testing at baseline, day 7 and day 28. Testing sessions consisted of a resting muscle biopsy of the vastus lateralis, body composition measurements, a graded exercise test on the cycle ergometer for VO2max and lactate threshold, and multiple Wingate tests for anaerobic exercise performance. Results showed all supplementation strategies increasing muscle carnosine levels over placebo after four weeks, but not between groups. Muscle creatine increased for all groups after four weeks, but not between groups. There were improvements for all groups with body composition after four weeks, despite the present study not including a specific training protocol. There were no group differences observed for aerobic exercise, blood lactate levels, lactate threshold, ventilatory threshold, peak power, mean power, total work or rate of fatigue. There were some trends for anaerobic exercise indicating groups supplementing with creatine may have greater improvements, however, these findings were not statistically significant. The present study failed to show any additive effects of beta-ALA and creatine supplementation for body composition, aerobic exercise, lactate threshold or anaerobic exercise measures. This could be due to the small sample size resulting in low power and effect sizes. Previous research has demonstrated that four weeks of beta-ALA and creatine supplementation was enough time to increase muscle carnosine and phosphagen levels. However, perhaps more time is needed for performance adaptations to occur, especially without the addition of an exercise training component.

Muscle fatigue has always been of vital importance in most sports. A few possible factors have been reported to be the cause of muscular fatigue during high intensity exercise; depletion of glycogen, oxidative stress, disruption of contractile mechanisms and accumulation of metabolites. One of the theories of the cause of muscular fatigue, both in endurance and intermittent sports, is decreased pH levels due to increased concentration of H+ ions dissociated from lactic acid in muscle. Carnosine, a fairly unnoticed ergogenic aid, taken in the form of β-alanine has shown to potentially delay the onset of fatigue. Supplementation of β-alanine, would increase carnosine levels in muscle and may counteract the decrease in pH since carnosine functions as a H+ buffer. The purpose of the present study was to examine the effects of 8 weeks supplementation of β-alanine in distance runners and Swedish division four soccer players on aerobic capacity, intermittent recovery and muscular fatigue. The runners (n = 15) were tested in lactate profiling tests and the soccer players (n = 22) were tested in the Yo-Yo intermittent endurance test pre and post the 8-week test-period. The yo-yo test did not result in significant difference between the soccer players’ β-group and control-group (p = 0,29). Neither did the lactate test result in significant differences between the distance runners’ β-group and control-group in any of the five variables measured. However, a trend in difference was seen between groups in both velocity at lactate threshold (VLT) (p = 0,11) and recovery blood lactate (RBL) (p = 0,14) where the β-group had increased slightly from 16,8 ± 1,6 km/h to 17,0 ± 1,2 km/h in VLT and decreased from 4,5 ± 1,6 mmol∙L-1 to 3,1 ± 1,0 mmol∙L-1 in RBL. The results suggested that β-alanine may delay the onset of fatigue and improve performance in endurance sports such as running by increasing the removal of lactate acid from muscle.

Mitochondrial and supernatant glutamate-oxalacetate transaminases (EC 2.6.1.1) and supernatant glutamate-pyruvate transaminase (EC 2.6.1.2) were purified 89, 204 and 240-fold respectively, from dolphin muscle. Starch gel electrophoresis of crude and purified perparations revealed that all three enzymes exist as single forms. Km values of a-ketoglutarate, alanine, pyruvate and glutamate for the glutamate-pyruvate transaminase were 0.45, 8.2, 0.87 and 15 mM, respectively. For the glutamate-oxalacetate transaminases, the Km values of a-ketoglutarate, aspartate, oxalacetate and glutamate were 0.76, 0.50, 0.10 and 9.4 mM, respectively, for the mitochondrial form and 0.13, 2.4, 0.06 and 3.2 mM, respectively, for the supernatant form. In all cases, as the assay pH was decreased from pH 7.3, the Km values of the a-keto acids decreased while those of the amino acids increased. This caused the apparent equilibrium constants for the glutamate-oxalacetate transaminases to remain independent of pH. These values were 9.2 and 6.8 for the mitochondrial and supernatant forms, respectively where K'eq = [asPartate][α-ketoglutarate]/[glutamate][oxalacetate]. Studies of the inhibition of the glutamate-oxalacetate transaminases by dicarboxylic acids indicated that these enzymes may be controlled by pools of metabolic intermediates. Three key roles are suggested for the transminases in the energy metabolism of the diving mammal. First, it is believed that a combined action of the transaminases could enhance energy production during hypoxia by providing (1) fumarate from aspartate for the ATP producing reversal of succinate dehydrogenase and (2) α-ketoglutarate from glutamate for the GTP producing succinyl thiokinase reaction. Next, diving mammals probably accumulate more NADH than other mammals during hypoxia. The glutamate-oxalacetate transaminases seem particularly well suited for restoring redox balance via the malate-aspartate cycle after aerobic metabolism is resumed. Finally, since migrating divers oxidize large amounts of stored fats, the combined reactions of the transaminases could be instrumental in providing increased supplies of oxalacetate to condense with the fat derived acetyl CoA in the citrate synthase reaction. / Science, Faculty of / Zoology, Department of / Graduate

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-14T13:38:53Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação (versão final) Leoncio.pdf: 3177730 bytes, checksum: 3ae8af6eb5ff1a7f234df372ba6107db (MD5) / Made available in DSpace on 2017-07-14T13:38:53Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação (versão final) Leoncio.pdf: 3177730 bytes, checksum: 3ae8af6eb5ff1a7f234df372ba6107db (MD5) Previous issue date: 2016-06-30 / FACEPE / Nos últimos anos, a crescente dificuldade para aquisição e transporte internacional de irradiadores de raios gama, como o irradiador de Co-60 GammaCell 220 da Nordion, levou ao desenvolvimento de irradiadores de altas taxas de dose com base em feixes de raios-X, que operam na faixa de 150kV. Uma das aplicações deste tipo de irradiador é a irradiação de insetos, de amostras de sangue, etc. Para estes procedimentos, é importante que se tenha dosímetros que possam monitorar os processos de irradiação. O objetivo deste trabalho é o estudo e caracterização da alanina para que possa ser aplicada em dosimetria de irradiadores de raios-X de altas taxas de dose. A alanina é um aminoácido com número atômico efetivo próximo ao do tecido humano e é utilizada para dosimetria por ressonância paramagnética eletrônica (RPE) para aplicações em radioterapia. Neste trabalho, foram produzidas pastilhas de alanina com diferentes aglutinantes, bem como pastilhas puras revestidas com duas camadas de verniz, para serem utilizadas como dosímetros. Foram utilizados como aglutinantes parafina, teflon e estearina em concentrações de 1 %, 2 % e 5 % da massa total das pastilhas. Testes mecânicos, como ultrassom e rot-up, foram realizados para avaliar a resistência mecânica das pastilhas produzidas. Após os testes mecânicos, verificou-se que as pastilhas preparadas com alanina pura apresentavam resistência mecânica suficiente para serem utilizadas como dosímetros. A reprodutibilidade de sua resposta RPE foi avaliada a partir da irradiação de dez dosímetros com dose de 100 Gy com uma fonte de Co-60. Para investigar a estabilidade da resposta da alanina-RPE com o tempo, três dosímetros foram irradiados com uma dose de 200 Gy com uma fonte de Co-60, sendo a leitura realizada após cinco horas da irradiação e releituras após 15, 40, 87, 110 e 137 dias da irradiação. Para obter as curvas de calibração para raios gama e raios – X, amostras foram irradiadas, respectivamente, com doses de 0 a 1 kGy e de 18,7 Gy a 206 Gy. Os resultados mostram que a resposta dos dosímetros de alanina-RPE apresentou reprodutibilidade com um coeficiente de variação de 1,2%. O estudo do desvanecimento da resposta dos dosímetros mostrou uma variação de 0,8% em 137 dias. De acordo com os resultados obtidos, o sistema de dosimetria alanina-RPE pode ser utilizado, de forma confiável, como um sistema padrão de transferência para as aplicações do irradiador de raios-X de baixa energia, o RS 2400. / In recent years, the increasing difficulty in acquisition and international transport of gamma ray radiators, as irradiator Co-60 Gammacell 220 of Nordion led to the development of high dose rate irradiators based on X-ray beams that they are operating in the 150kV range. One application of this type of irradiator and irradiation of insects, blood samples, etc. For these procedures, it is important to have dosimeters that can monitor the irradiation processes. The objective of this work is the study and characterization of alanine that can be applied in dosimetry irradiating X-ray of high dose rates. Alanine is an amino acid in effective atomic number close to that of human tissue and is used for dosimetry for electron paramagnetic resonance (EPR) for applications in radiotherapy. In this work, alanine pellets were produced with different binders as well as pure tablets coated with two coats of varnish to be used as dosimeters. They were used as binders paraffin, teflon and stearin in concentrations of 1%, 2% and 5% of the total mass of the pastilles. Mechanical tests, such as ultrasound and rot-up were conducted to evaluate the mechanical strength of the tablets produced. After mechanical testing, it was found that the tablets prepared with pure alanine had sufficient mechanical strength to be used as dosimeters. The reproducibility of EPR response was evaluated from the irradiation ten dosimeters with dose 100 Gy with a source of Co-60. To investigate the stability of alanine EPR response over time of three dosimeters were irradiated with a dose of 200 Gy with a source of Co-60, the reading being performed after five hours of irradiation and readings after 15, 40, 87, 110 and 137 days of irradiation. To obtain the calibration curves for gamma rays and X - ray irradiated samples were, respectively, at doses from 0 to 1 kGy and 18.7 Gy to 206 Gy. The results show that the response of EPR-alanine dosimeters showed reproducibility with a coefficient of variation 1.2%. The study fading dosimeters response showed a variation of 0.8% in 137 days. According to the results, the system alanine EPR dosimetry can be used reliably as a pattern transferring system for irradiating the applications of low-energy X-rays, the RS 2400.

A convenient, economical, large scale synthesis of N-acetyldehydro-alanine (a-acetamidoacrylic acid) and its methyl ester was developed via a sequence of N-chlorination-dehydrochlorination. The method was extended to the synthesis of the corresponding N-benzoyl, N-phenylacetyl and N-benzyloxycarbonyl derivatives. The conversion of a-methoxy-N-acetyl alanine derivatives to the corresponding a-mercapto alanine derivatives, using zinc chloride and an appropriate mercaptan, was investigated. Methyl a-methoxy-N-acetyl-D, L-alaninate was successfully converted to the a-acetylthio derivative, in 24% yield; however, a 90% yield could be obtained by treatment of the dehydroalanine derivative with hydrogen chloride gas in neat thiolacetic acid. A facile conversion of the a-acetylthio derivative to the a-methoxy derivative, using sodium methoxide in methanol, was observed to occur. The normally facile conversion of an acetylthio group to a mercapto group, using sodium borohydride, gave mixtures of the a-mercapto derivative and alanine derivative. The reactions at the a position of a-hetero-N-acetyl-D,L-alanines and a,B-disubstituted N-acetyl-D,L-alanine derivatives seemed consistent with the formation of an acylimine intermediate under basic conditions and a carbonium ion intermediate under acidic conditions. From these studies, a facile, clean synthesis of a-halo-a-mercapto-and a-alkoxy-N-acetyl-D,L-alanine derivatives was accomplished. All attempts to synthesize a,B-dimercapto derivatives failed because the B-halogen could not be replaced with a mercapto group when the a position was a mercapto or methoxy derivative. Attempts to generate a B-mercapto-a-halo derivative also failed. A facile synthesis of the E and Z isomers of methyl B-chloro-N-acetyldehydroalaninate was developed. The ratio of Z to E isomers was found to vary with the base used for the elimination. The E and Z isomers of methyl B-chloro-N-acetyldehydroalaninate were converted to the B-mercapto derivatives by reaction with mercaptan. The reaction proceeded with retention of stereochemistry. The formation of B-substituted N-acetyldehydroalanine derivatives seemed consistent with an acylimine intermediate followed by a sequence of Michael-type addition and dehydrochlorination. A study of the conversion of the B-mercapto-N-acetyldehydroalanine derivatives to mixed dithioacetals, which would be useful in the synthesis of natural antibiotics, was undertaken. This approach to the synthesis of mixed dithioacetals was unsuccessful because an exchange of mercapto groups was observed, the addition of a second, different mercaptan failed or the yield was too low to be synthetically useful. A synthesis of B,B-dimercapto-N-aeetyldehydroalanine (mixed, unsaturated dithioacetals) derivatives was accomplished. However, because of low yields, a reduction to B,B- dimercapto-N- acetyl-D,L-alanine derivatives (mixed dithloacetals) was not investigated.

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