Thesis (Ph. D.)--University of Montana, 2007. / Title from title screen. Description based on contents viewed Aug. 20, 2007. Includes bibliographical references (p. 116-120).

In the first phase of the current study, metabolic combinatorial solutions were in ovo administered in broiler eggs on Day 18 of incubation to investigate their effects in broiler chick tissue nutrient profiles until Day 10 of posthatch growout. In the second phase, the effects of in ovo injection of L-carnitine on Day 18 of incubation in broiler eggs were examined. The treatment solutions used in these studies were considered to play significant roles in various biochemical pathways, and were hence tested to determine whether they could potentiate the physiological growth and development of the broiler embryos and posthatch chicks. The in ovo injection of treatment solutions in both trials did not produce any significant increase in performance or slaughter yield in broiler chicks. However, positive trends were determined for rate of hatch and tissue nutrient profiles, which implied that the in ovo administration of nutrient supplements may be supportive of embryonic development and posthatch growout performance.

Metabolic flexibility plays a significant role in energy homeostasis by regulating fuel selection in correspondence to energy demand. Obese and type II diabetic populations have displayed a hindered ability to properly transition from fat oxidation while in a fasted state to carbohydrate oxidation once fed, leading to a buildup of mitochondrial metabolites such as acylcarnitines. Carnitine, essential for fatty acyl-CoA transport through the inner and outer mitochondrial membranes, can be an indicator of mitochondrial distress as elevated levels tend to spill over into plasma suggesting a disruption in oxidation. The current study was designed to examine the effect of short term, high fat feeding on plasma acylcarnitine species diversity and levels and if acylcarnitines are associated with metabolic flexibility. 13 healthy, non-obese, sedentary males, aged 18-40 years participated in this study. Following a 12-hour overnight fast a biopsy was taken from the quadricep before and 4 hours after a high fat meal. Blood draws were obtained pre-biopsy while fasted and every hour for 4 hours post high fat meal consumption. Acylcarnitines from plasma were converted to their butyl esters and analyzed by electrospray ionization tandem mass spectrometry (MS/MS). Changes were observed in acetylcarntine (P=0.0125), glucose oxidation (P=0.0295), C16:1/C16:0 desaturation index (P= 0.0397), and C18:1/C18:0 desaturation index (P=0.0012). We did not find that individual changes in flexibility correlated with circulating acylcarnitine measurements in a fasted state / Master of Science

Doctor of Philosophy / Department of Animal Sciences and Industry / Jim Nelssen / A total of 701 pigs were used to evaluate effects of natural vitamin E relative to synthetic vitamin E in sow diets, late gestation feeding level on sow reproductive performance, dietary L-carnitine and chromium on sow reproductive performance, and experimental design on nursery pig trial interpretation. As D-α-tocopheryl acetate increased in the sow’s diet, concentrations of α-tocopherol increased (P < 0.03) in sow plasma, colostrum, milk, pig plasma, and pig heart. Regression analysis indicated that the bioavailability coefficients for D-α-tocopheryl acetate relative to DL-α-tocopheryl acetate ranged from 2.1 to 4.2 for sow and pig plasma α-tocopherol, 2.9 to 3.0 for colostrum α-tocopherol, 1.6 for milk α-tocopherol, 1.8 for heart α-tocopherol, and 2.0 for liver α-tocopherol. Overall, this study indicates that the relative bioavailability for D-α-tocopheryl acetate relative to DL-α-tocopheryl acetate varies depending on the response criteria but is greater than the standard potency value of 1.36. Increasing sow gestation feeding level by 0.9 kg from d 90 of gestation through farrowing reduced (P = 0.001) daily lactation feed intake in gilts, but also resulted in improved conception rate in gilts, whereas increasing late gestation feeding level decreased conception rate in sows (interaction; P = 0.03). Increasing late gestation feed intake in gilts also increased (P < 0.02) pig weaning weights during the second parity. Increasing late gestation feeding levels did not improve performance of older sows. Adding L-carnitine and chromium from chromium picolinate to sow gestation and lactation diets reduced (P = 0.01) the amount of sow weight loss during lactation, however, did not improve (P > 0.05) litter size, pig birth weight, or the variation in pig birth weight. Blocking pens of nursery pigs by BW in a randomized complete block design (RCBD) did not improve the estimates for σ2error compared to a completely randomized design (CRD) where all pens were allotted to have similar means and variations of body weight. Therefore, the added degrees of freedom for the error term in the CRD allowed more power to detect treatment differences for the CRD compared to the RCBD.

<p>Acetyl-CoA holds a prominent position as the common metabolic intermediate of glucose, amino acid and fatty acid oxidation. Because acetyl-CoA fuels the tricarboxylic acid (TCA) cycle, the primary source of reducing equivalents that drives mitochondrial oxidative phosphorylation, understanding acetyl-CoA pool regulation becomes imperative to understanding mitochondrial energetics. Carnitine acetyltransferase (CrAT), a muscle-enriched mitochondrial enzyme, catalyzes the freely reversible conversion of acetyl-CoA to its membrane permeant carnitine ester, acetylcarnitine. Because CrAT has long been thought to regulate the acetyl-CoA metabolite pool, we investigated the role of CrAT in acetyl-CoA regulation. Although the biochemistry and enzymology of the CrAT reaction has been well studied, its physiological role remains unknown. Investigations herein suggest that CrAT-mediated maintenance of the mitochondrial acetyl-CoA pool is imperative for preservation of energy homeostasis. We provide compelling evidence that CrAT is critical for fine-tuning acetyl-CoA balance during the fasted to fed transition and during exercise. These studies suggest that compromised CrAT activity results in derangements in mitochondrial homeostasis.</p><p>In chapter 3, we examined the effects of obesity and lipid exposure on CrAT activity. Recent studies have shown that acetyl-CoA-mediated inhibition of pyruvate dehydrogenase (PDH), the committed step in glucose oxidation, is modulated by the CrAT enzyme. Because PDH and glucose oxidation are negatively regulated by high fat feeding and obesity, we reasoned that nutritional conditions that promote lipid availability and fat oxidation might likewise compromise CrAT activity. We report an accumulation of long chain acylcarnitines and acyl-CoAs but a decline in the acetylcarnitine/acetyl-CoA ratio in obese and diabetic rodents. This reduction in the skeletal muscle acetylcarnitine/acetyl-CoA ratio was accompanied by a decrease in CrAT specific activity, despite increased protein abundance. Exposure to long chain acyl-CoAs in vitro demonstrated that palmitoyl-CoA acts as a mixed model inhibitor of CrAT. Furthermore, primary human skeletal muscle myocytes exposed to fatty acid and or CPT1b overexpression had elevated long chain acylcarnitines but decreased production and efflux of CrAT-derived short chain acylcarnitines. These data suggest that exposure to fatty acids in obesity and diabetes can counter-regulate the CrAT enzyme leading to decreased activity. </p><p>Alternatively, chapter 4 addresses the importance of acetyl-CoA buffering during exercise and suggests that a deficit in CrAT activity leads to fatigue. Because CrAT is highly expressed in tissues specifically designed for work and because acetylcarnitine, the primary product of the CrAT reaction, is increased during contraction, we reasoned that CrAT could play an important role in exercise. To investigate this possibility, we employed exercise intervention and ex-vivo analysis on a genetically novel mouse model of skeletal muscle CrAT deficiency (CrATSM-/-). Though resting acetyl-CoA levels were elevated in CrATSM-/- mice, these levels dropped significantly after intense exercise while acetylcarnitine content followed the opposite pattern. This contraction-induced acetyl-CoA deficit in CrATSM-/- mice was coupled with compromised performance and diminished whole body glucose oxidation during high intensity exercise. These results imply that working muscles clear and consume acetylcarnitine in order to maintain acetyl-CoA buffering during exercise. Importantly, provision of acetylcarnitine enhanced force generation, delayed fatigue and improved mitochondrial energetics in muscles from CrATfl/fl controls but not CrATSM-/- littermates, emphasizing the importance of acetyl-CoA maintenance. In aggregate, these data demonstrate a critical role for CrAT-mediated acetyl-CoA buffering in exercise tolerance and suggest its involvement in energy metabolism during skeletal muscle contraction and fatigue. These findings could have important clinical implications for individuals with muscle weakness and fatigue due to multiple conditions, such as peripheral vascular or cardiometabolic disease. </p><p>In summary, data herein emphasize the role of CrAT in regulation of mitochondrial acetyl-CoA pool. We demonstrate that CrAT is critical for fine-tuning acetyl-CoA balance both during the fasted to fed transition and during exercise. These data suggest that a deficit in CrAT activity leads to glucose intolerance and exercise fatigue. We examine these studies and suggest future areas of study.</p> / Dissertation

Thesis (MPhil)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: 60-day growth experiment was conducted to investigate the effect of dietary Lcarnitine supplementation on the production performance parameters of Mozambique tilapia, Oreochromis mosambicus. A number of approximately 140 tilapia fry with average weight of l.4g ± 0.71g were stocked in each of 40 fine-meshed hapas (I mx 1mx 1.5m) submerged within a complete recirculation pond system. During the first 30 days of the experiment water temperatures ranged from 19 to 23°C where after it decreased to 16-20°C for the consecutive 30-day period. Dietary treatments consisted of 8 replicates of 5 levels of L-carnitine supplementation labelled as Co, C250,C500, C750 and C 1000represented Omg, 250mg, 500mg, 750mg and 1000mg L-carnitine supplementation per kg feed respectively. Results were analyzed for significant differences using one-way analysis of variance (ANOVA) and Tukey's pairwise comparison test for growth rate, feed intake (FI) and feed conversion efficiency. After completion of the trial 8 fish from each hapa were sacrificed and analyzed for cephalosomatic index (CSI), dress out percentage (viscera, gills and head excluded), viscerosomatic index (VSI) and hepatosomatic index (HSl). Poor production performance results were generally observed as water temperatures were sub-optimal, especially during the second 30-days period. Results from the trial indicate no significant differences (P>0.05) between treatments for weight gain, FCR, FI and VS!. A negative trend was observed for FCR with increasing level of L-carnitine supplementation for both the first 30-day period (1.50±0.07, 1.53±0.08, 1.58±0.09 and 1.61±0.17 for C250,C50Q,C750and C 1000)as well as for the consecutive lower temperature 30-day period (2.22±0.10, 2.25±0.ll, 2.27±0.28 and 2.29±0.2l for C250, C500, C750 and C 10(0)'Although statistically not significant, fish fed the C250showed better performance in dress out percentage weight either than the control or the higher levels. The increasing trend for head weight with increasing level of L-carnitine supplementation were significant (P<0.05) from Co and C250with and above C500.The decreasing trend for liver weight with increasing level of L-carnitine supplementation became significant (P<0.05) with and above C750.The results of the current study showed a trend in the improvement of L-carnitine on the production performance parameters. However, the natural content of L-carnitine in the basal diet impaired with the inclusion levels, thus further research at lower inclusion levels is recommended. / AFRIKAANSE OPSOMMING: 'n Proef oor 'n tydperk van 60-dae is onderneem om die effek van L-karniten aanvulling op produksie prestasie parameters van Mosambiek tilapia (0. mosambicusi te ondersoek. 140 tilapia vingerlinge met 'n gemiddelde massa van lAg ± 0.7lg is ewekansig uitgeplaas in 40 eksperimentele hapa-hokkies (lmxlmx1.5m) in "n hersirkulasie sementdam-stelsel. Gedurende die eerste 30 dae van die proef het water temperatuur gewissel tussen 19 to 23°C waarna dit gedaal het na tussen l6-20°C vir die opeenvolgende 30-dag periode. Proef-rantsoen behandelings het bestaan uit 8 herhalings van 5 vlakke van L-karnitien aanvulling, naamlik Co,C250, C500, C750 en CIOOOvir Omg, 250mg, 500mg, 750mg en 1000mg L-karnitien aanvulling per kg voer afsonderlik. Resultate was ontleed vir betekenisvolle verskille deur gebruik te maak van analise van variansie (ANOVA) ontleding en die Tukey se vergelykende toets vir groeitempo, voerinname en voeromsettingsverhouding. Aan die einde van die proefperiode is 8 visse van elke hapa ontleed vir liggaamskomponent-samestelling (kop-, ingewande- en hepatosomatiese indekse. Ondergemiddelde produksie resultate is waargeneem wat toegeskryf kan word aan onder-optimale water temperature, veral gedurende die tweede 30-dag periode van die proef. Proef resultate het geen betekenisvolle verskille (P>0.05) in massatoename, voeromsettingsverhouding (VOV) of visserosomatiese indeks tussen behandelings getoon nie. 'n Negatiewe neiging is waargeneem vir VOV met toenemende vlakke van L-kamitien insluiting vir beide die eerste 30 dag periode (1.50±0.07, 1.53±0.08, 1.58±0.09 and 1.61±0.17 for C250, C500, C750 and CIOOO) sowel as vir die opvolgende 30-day periode nie (2.22±0.10, 2.25±0.11, 2.27±0.28 and 2.29±0.21 for C250, C50o, C750 and CIOOO). 'n Toenemende neiging vir kop-massa met toenemende L-kamitien insluiting was betekenisvol (P<0.05) vanaf Co en C250 met en hoër as C500. 'n Dalende neiging vir lewermassa met toenemde L-kinsluiting was betekenisvol (P<0.05) met en hoër as C750. Resultate van die proef dui oor die algemeen op 'n neiging tot verbeterde produksie prestasie parameters van tilapia vingerlinge met toenemde insluiting van Lkamitien. Verdere navorsing word aanbeveel om die invloed van natuurlike Lkamitien in die proteïen-bronne van die basaalrantsoen te op die gebrek aan betekenisvolheid van hierdie neiging te verklaar.

Organic chemistry is a valuable tool for studying enzyme mechanisms. Upon incubation with a specific enzyme, synthetic substrate analogues labeled with heavy atoms or carrying extra functional groups can provide mechanistic insights. In the present work, new compounds were synthesised in order to study the mechanism and substrate selectivity of two enzymes: human γ-butyrobetaine hydroxylase and bacterial carboxymethylproline synthase. γ-Butyrobetaine hydroxylase (BBOX) is an Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase that catalyses the stereospecific hydroxylation of γ-butyrobetaine, the final step of L-carnitine (L-Car) biosynthesis in mammals. Substrate analogues were synthesised to probe BBOX specificity in vitro. Some of those unnatural substrates were oxidised by BBOX and the products identified using a range of analytical techniques. 3-(2,2,2-Trimethylhydrazinium)propionate (THP) is a clinically used BBOX inhibitor. Under standard assay conditions, THP was oxidised by BBOX. NMR studies have identified the products of this reaction to be malonic acid semialdehyde, formaldehyde, dimethylamine and 3-amino-4-(methylamino)butanoic acid. The formation of 3-amino-4-(methylamino)butanoic acid suggests that BBOX can catalyse a Stevens type rearrangement involving N-N bond cleavage and C-C bond formation. The proposed structures and mechanisms were confirmed by mass spectrometric and NMR analyses using [¹³C]-labeled THP as well as synthetic standards of both enantiomers of 3-amino-4-(methylamino)butanoic acid. Although the structure of the rearrangement product was confirmed, the stereochemistry remains unknown. Altogether, these studies revealed the unprecedented nature of a BBOX-catalysed C–C bond formation reaction upon THP oxidation and may inspire the design of improved inhibitors for BBOX and other 2OG oxygenases. Pectobacterium carotovorum CarB and Streptomyces cattleya ThnE are two carboxymethylproline synthases (CMPS) that catalyse an early step in carbapenem antibiotics biosynthesis. CMPS produces (2S,5S)-carboxymethylproline (t-CMP) from malonyl-CoA and L-glutamate semi-aldehyde. L-Glutamate semi-aldehyde exists in equilibrium with L-5-hydroxyproline and L-pyrroline-5-carboxylate in solution (collectively abbreviated L-GHP). Because of the high stereoselectivity of t-CMP formation and the growing interest in novel carbapenem antibiotics, CMPS is potentially an interesting biocatalyst. A series of L-GHP analogues were synthesised and tested as CMPS substrates in an attempt to produce unnatural t-CMP derivatives enzymatically. Methyl-substituted L-GHP analogues were accepted by CMPS and the t-CMP products could be further carried through to the corresponding bicyclic carbapenams using CarA, a β-lactam synthetase. These results demonstrate the versatility of the early carbapenem biosynthetic pathway and the possibility of introducing structural diversity using synthetic substrate analogues. A crystal structure of S. cattleya ThnE was obtained in complex with L-proline and coenzyme A, giving the first insight into substrate binding. This structural information will potentially allow further rational mutagenesis studies aiming to broaden the range of unnatural L-GHP analogues accepted by CMPS.

Pseudomonas aeruginosa is found in numerous environments and is an opportunistic pathogen affecting those who are immunocompromised. Its large genome encodes tremendous metabolic and regulatory diversity that enables P. aeruginosa to adapt to various environments. We are interested in how P. aeruginosa senses and responds to the host-derived compounds, carnitine and acylcarnitines. Acylcarnitines can be hydrolyzed to carnitine, where the liberated carnitine and its catabolic product glycine betaine can be used as osmoprotectants, for induction of the virulence factor phospholipase C, and as sole carbon, nitrogen, and energy sources. P. aeruginosa is incapable of de novo synthesis of carnitine and acylcarnitines and therefore imports these compounds from exogenous source. Short-chain acylcarnitines are imported by the ABC transporter CaiX-CbcWV. Medium- and long-chain acylcarnitines are hydrolyzed extracytoplasmically and the liberated carnitine is transported through CaiX-CbcWV. Once in the cytoplasm, short-chain acylcarnitines are hydrolyzed by the L-enantiomer specific hydrolase, HocS. The transcriptional regulator CdhR is divergently transcribed from the carnitine catabolism operon and we have identified the upstream activating region, the binding site sequence, and essential residues required for CdhR binding and induction of the carnitine operon. Carnitine catabolism is repressed by glucose and glycine betaine at the transcriptional level. Furthermore, using two different cdhR translational fusions we show that CdhR enhances its own expression and that GbdR, a related transcription factor, contributes to cdhR expression by enhancing the level of basal expression. These studies are the first to determine the mechanism of O-acylcarnitine transport, metabolism, and the regulation of these processes, which contribute to utilization of these compounds for P. aeruginosa survival in diverse environments.

A dissertation submitted to the Faculty of medicine of the university of Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Masters of science Johannesburg 1991 / Carnitine plays a role in the transport of activated long chain fatty acyl groups from the site of activation to the site of beta-oxidation in the mitochondria. Endogenous synthesis of carnitine from the amino acids methionine and lysine, takes place mainly in the liver. From there free and acyl carnitine are released into the blood and transported to other tissues.A few studies have indicated that poor nutritional status may lead to carnitine deficiency.[Abbreviated Abstract. Open document to view full version] / GR2017

A subnutrição é uma das comorbidades mais freqüentes que atinge os pacientes oncológicos. Entender as conseqüências do câncer nas alterações do metabolismo energético é necessário para o estabelecimento de estratégias que previnam o desenvolvimento e tratem a má-nutrição. A carnitina desempenha papel fundamental no metabolismo energético lipídico. Sendo que o objetivo deste estudo foi avaliar os níveis plasmáticos nos pacientes com câncer no pré cirúrgico e relacionar com os resultados da história alimentar, antropometria, impedância bioelétrica, calorimetria indireta, aminoacidemia e valores urinários de carnitina e nitrogênio. Trata-se de um trabalho prospectivo no qual foram escolhidos aleatoriamente 4 grupos, sendo um de pacientes portadores de câncer esofágico ou gástrico (n=24), e os outros 3 considerados controles. O primeiro grupo controle foi o obesos (n=16), o segundo de voluntários saudáveis (n=12), enterectomizados (n=6) Os valores médios de carnitinemia , em todos os grupos, variou entre 60 e 80 µM no plasma e urinária entre 78 e 124 µM, sem diferenças estatísticas entre os grupos. Quando avaliados os níveis de carnitina plasmático, 80% (p<0.05) dos pacientes com câncer apresentaram deficiência associados à excreção urinária inferior a 5 mol/kg/dia, consumo insuficiente de proteínas e baixa reserva adiposa. No entanto os níveis de metionina e lisina, bem como o gasto energético de repouso não apresentaram diferença com os controles. A deficiência de carnitina nestes pacientes pode comprometer o metabolismo energético além de estar associado à ocorrência de fadiga e piora da qualidade de vida. / The malnutrition is one most common comorbidity among hospitalized patients. Understanding cancer consequences in energetic metabolim changes is necessary in order to avoid malnutrition or to treat it. Carnitine has a important role in lipid metabolism. The aim of this study was to evaluate the levels of serum carnitine in patients with stomach and esophagus cancer and correlated with dietary intake, body composition, resting energy expenditure, carnitine and amino acid serum levels and urinary excretion of carnitine. Twenty-four cancer patients were assessed. Cancer patients were compared with obese (n=16), healthful (n=12) and short bowel disease (n=6). The mean values of serum carnitine and urinary carnitine among all groups were 60-80 µM and 74-124 µM respectively. Serum carnitine levels between cancer patients and other groups were significantly different. 80% of cancer patients had low serum levels, which was associated with urinary below 5 µmol/Kg/day, decreased protein and low adipose tissue. However, the methionine and lysine levels, as well as the resting energy expenditure had no difference when compared with the healthy volunteers. Carnitine deficiency in cancer patients can affect energetic metabolism and contribute to the progression of cachexia.