A doença da urina do xarope do bordo (MSUD) é causada pela deficiência na atividade do complexo da desidrogenase dos U-cetoácidos de cadeia ramificada (BCKAD), promovendo o acúmulo dos aminoácidos de cadeia ramificada (BCAA) leucina (Leu), isoleucina (Ile) e valina (Val) e seus U-cetoácidos correspondentes (BCKA). A MSUD caracteriza-se por cetoacidose, ataxia, coma, retardo mental e psicomotor. Estudos em animais demonstraram que BCAA e BCKA estimulam a lipoperoxidação e reduzem capacidade antioxidante cerebral em ratos. Também há evidências de que o estresse oxidativo ocorra em pacientes com MSUD no diagnóstico e durante o tratamento e que devido à terapia com dieta restrita e hipoproteica eles possuam deficiência de L-carnitina (L-car), um importante composto para o metabolismo energético. Recentemente, estudos demonstraram o papel antioxidante e anti-inflamatório da L-car, através de sua ação antiperoxidativa, sequestradora de espécies reativas e efeito estabilizador de danos às membranas celulares. Considerando que a fisiopatologia da MSUD ainda é pouco compreendida e que existe um crescente número de estudos enfatizando o envolvimento do estresse oxidativo na doença, neste trabalho foi investigado o efeito in vitro e in vivo da L-car sobre o estresse oxidativo e o dano inflamatório na MSUD tendo como objetivos: A) estudar a indução ao dano oxidativo pelos metabólitos acumulados na MSUD, verificando o possível papel antioxidante da L-car sobre o dano ao DNA in vitro; B) avaliar o efeito in vivo da suplementação de 50 mg/kg/dia de L-car sobre: b.1) a indução do dano ao DNA em leucócitos de pacientes com a MSUD tratados com dieta de restrição proteica, correlacionando as concentrações dos principais metabólitos acumulados nesta doença e verificando o possível papel antioxidante da suplementação da Lcar; b.2) a concentração de citocinas pró-inflamatórias em plasma de pacientes com MSUD tratados com dieta de restrição proteica e a correlação com o estresse oxidativo; b.3) os parâmetros de dano oxidativo à biomoléculas em urina de pacientes com MSUD sob dieta de restrição proteica; C) avaliar o efeito da L-car sobre o estresse oxidativo causado pelos metabólitos acumulados na MSUD em córtex cerebral e cerebelo de ratos Wistar, através de um modelo crônico de indução química da doença. Verificou-se que a Leu e o seu - cetoácido correspondente, o ácido -cetoisocapróico (KIC), causaram danos ao DNA in vitro e L-car foi capaz de diminuir significativamente essas alterações, principalmente as causadas pelo KIC. Quando testado o efeito da suplementação de L-car sobre o dano ao DNA em pacientes MSUD, observou-se um aumento significativo de lesões ao DNA em pacientes com dieta de restrição proteica quando comparados aos controles e a terapia com L-car foi capaz de diminuir significativamente os níveis desses danos. Também foram verificadas correlações do tipo negativa entre as concentrações de L-car e os índices de dano ao DNA e do tipo positiva entre as lesões ao DNA e níveis de MDA, marcador de lipoperoxidação, explicitando uma relação entre o dano ao DNA observado nos pacientes com MSUD, estresse oxidativo e o benefício da suplementação de L-car. Também averiguou-se o efeito da terapia de L-car sobre as citocinas pró-inflamatórias interleucina 1Y (IL-1Y), interleucina 6 (IL-6) e interferon gama (INF- Z). Constatou-se aumentos significativos de IL-1Y, IL-6 e INF- Z no plasma de pacientes com MSUD antes da suplementação de L-car e uma reversão completa desses valores aos níveis dos controles para IL-1Y e INF- Z após a administração de L-car. Ainda, verificou-se que a L-car pode auxiliar na defesa celular contra a inflamação e o estresse oxidativo, observando-se uma correlação negativa entre todas citocinas testadas e as concentrações de L-car, e uma correlação positiva entre o conteúdo de MDA e níveis de IL-1Y e IL-6. Constatou-se também que as medidas de di-tirosina (dano oxidativo a proteínas) e isoprostanos (dano de lipoperoxidação) estavam aumentadas e a capacidade antioxidante total diminuída na urina de pacientes com MSUD sem terapia com L-car e a suplementação deste composto induziu efeitos benéficos sobre estes parâmetros, reduzindo os níveis de di-tirosina e isoprostanos e aumentando a capacidade antioxidante medida em urina. Foi também observado um aumento de KIC urinário após dois meses de tratamento com L-car, quando comparado com o grupo controle, demonstrando um incremento da excreção deste metabolito tóxico. Desta forma, esses resultados sugerem um efeito de reversão de dano oxidativo pela L-car e que a urina pode ser utilizada para monitorar este tipo de lesão em pacientes afetados pela MSUD. Por fim, foram analisados em córtex cerebral e cerebelo de ratos Wistar submetidos ao modelo crônico de MSUD: espécies reativas ao ácido tiobarbitúrico (TBARS), para avaliar lipoperoxidação, conteúdo de carbonilas (dano oxidativo proteico), oxidação de diclorofluoresceína (DCF), para quantificar produção de espécies reativas teciduais, conteúdo de glutationa reduzida (GSH) que é um importante antioxidante não enzimático e a atividade das enzimas antioxidantes catalase (CAT), superóxido dismutase (SOD), glutationa peroxidase (GPx) e glicose-6-fosfato-desidrogenase (G6PD). Os resultados mostraram que a administração crônica de BCAA estimulou a lipoperoxidação, o dano oxidativo proteico, aumento de espécies reativas e diminuição das defesas antioxidantes enzimáticas e não enzimáticas, especialmente em córtex cerebral e o tratamento com L-car foi capaz de prevenir estes efeitos, exceto o dano oxidativo a proteínas. Em conjunto, estes resultados demonstram que os metabólitos acumulados na MSUD induzem dano oxidativo a biomoléculas (lipídios, proteínas e DNA), diminuem o status antioxidante e promovem aumento de processos inflamatórios. Ainda, estes dados podem contribuir para a compreensão dos mecanismos de ação dos efeitos citotóxicos dos metabólitos acumulados na MSUD e evidenciar o papel do estresse oxidativo e da inflamação na neuropatofiosiologia desta doença, além do efeito protetor da L-car sobre este processo. O estudo de antioxidantes, como a L-car, pode propor uma abordagem terapêutica adicional ao que é empregado atualmente para pacientes com MSUD, que é essencialmente dietética e, portanto, de difícil manejo. / Maple syrup urine disease (MSUD) is caused by deficiency of the activity of the mitochondrial enzyme complex branched-chain U-ketoacid dehydrogenase (BCKAD). The metabolic defect leads to accumulation of the branched chain amino acids (BCAA) leucine (Leu), isoleucine (Ile) and valine (Val) and the corresponding branched-chain U-keto acids. The clinical features of MSUD include ketoacidosis, seizures, coma, psychomotor delay and mental retardation. Treatment consists in Leu, Val and Ile restricted diet. Studies in animals have demonstrated that lipid peroxidation is stimulated by BCAA and BCKA in brain of rats and these metabolites reduce in vitro and in vivo the cerebral capacity to modulate the damage associated to increased free radical production. Also, there is evidence that oxidative stress occurs in MSUD patients at diagnosis and during treatment and that due to terapy with protein restricted diet they present L-carnitine (L-car) deficiency, an important compound for energy metabolism. Recent studies have demonstrated the antioxidant and anti-inflammatory role of L-carnitine (L-car), through its action against peroxidation in different tissues by various mechanisms, a scavenger of reactive oxygen species and the stabilizing effect of damage to cell membranes. Considering that the pathophysiology of MSUD is still poorly understood, and that there is an increasing number of studies emphasizing the oxidative stress involvement in the disease, this study investigated the in vitro and in vivo effect of L-car on oxidative stress and inflammatory damage in MSUD with the following purposes: A) to study the induction of damage by accumulated metabolites in MSUD, analyzing the possible antioxidant role of L-car on DNA damage in vitro; B) to evaluate the in vivo effect of 50 mg/kg/day of L-car supplementation about: b.1) the induction of DNA damage in leukocytes of MSUD patients treated with protein-restricted diet, correlating this damage with the concentrations of the major metabolites accumulated in this disorder and checking the possible antioxidant role of L-car supplementation; b.2) plasma inflammatory cytokines in treated MSUD patients with protein-restricted diet and the correlation with oxidative stress; b.3) oxidative damage parameters in urine of MSUD patients with protein-restricted diet supplemented with L-car; C) to investigate the BCAA effect on some oxidative stress parameters and evaluate the L-car efficacy against these possible pro-oxidant effects in cerebral cortex and cerebellum of rats submitted to a chronic chemically-induced model of MSUD. DNA damage index (DI) showed that Leu and -ketoisocaproic acid (KIC) groups was significantly higher than that of the control group, and that L-car was able to significantly prevent this damage, especially that due to KIC. Accordingly, DNA DI in MSUD patients under BCAA-restricted diet was significantly increased as compared to controls and L-car supplementation was able to significantly decrease this parameter. It was also verified a significant positive correlation between DNA DI and MDA content, a marker of lipid peroxidation. Furthermore, we found an inverse significant correlation between DI and L-car levels. These results strengthen a relationship between DNA damage observed in MSUD patients, oxidative stress and the L-car supplementation benefit. The role of L-car on plasma inflammatory cytokines interleukin-1Y (IL-1Y), interleukin-6 (IL-6) and interferon-gamma (INF- Z) was also evaluated in these patients. Significant increases of IL-1Y, IL-6, and INF- Z were observed before the treatment with L-car. Moreover, there is a negative correlation between all cytokines tested and L-car concentrations and a positive correlation among the MDA content and IL-1Y and IL-6 values after L-car supplementation. It was also demonstrated that the oxidative stress parameters di-tyrosine (oxidative protein damage) and isoprostanes (lipid peroxidation assay) were increased and the antioxidant capacity was reduced in urine of MSUD patients without L-car therapy and that the supplementation of this compound induced beneficial effects on these parameters, so reducing the di-tyrosine and isoprostanes levels and increasing the antioxidant capacity. It was also showed a significant increase in urinary KIC after 2 months of L-car treatment compared to control group, demonstrating an increased excretion of this toxic metabolite. In conclusion, these results suggest a reversion effect of the oxidative damage by L-car and that urine can be used to monitorize oxidative damage in patients affected by this disease. The following parameters were analysed in cerebral cortex and cerebellum of Wistar rats submitted to MSUD chemically-induced chronic model: thiobarbituric acid reactive species (TBA-RS), to evaluate lipid peroxidation, carbonyl content to evaluate protein oxidative damage, DCF oxidation to quantify reactive species production, reduced glutathione (GSH), an important non-enzymatic antioxidant and the activities of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glucose-6-phosphate dehydrogenase (G6PD). The results showed that the chronic administration of BCAA was able to promote both lipid and protein oxidation, increase of reactive species production and decreased brain antioxidant defenses, especially in cerebral cortex and that L-car was able to prevent these effects, except for oxidative damage to proteins. Taken together, these results demonstrate that the metabolites accumulated in MSUD cause oxidative damage to biomolecules (lipids, proteins and DNA), decrease antioxidant status and promote increased inflammatory processes. These results may contribute to the understanding of the mechanism of action of the cytotoxic effect of the metabolites accumulated in MSUD and the role of oxidative stress and inflammation in the MSUD neuropathophysiology besides the protective effect of L-car on this process. The study of antioxidants like L-car can opens an additional therapeutic approach to that currently employed for MSUD patients, which is primarily dietary and therefore difficult to handle.

Doctor of Philosophy / Department of Animal Sciences and Industry / Bradley J. Johnson / We used a total of fifty-nine gilts (BW=137.7 kg) from three different breeding groups were used to assess the effects of feeding L-carnitine during gestation on gilt growth characteristics, gilt and fetal blood metabolites, litter characteristics, and IGF axis components in fetal hepatic and skeletal muscle, maternal uterine and chorioallantois tissues, and porcine embryonic myoblasts collected from fetuses. Experimental treatments were arranged in a 2 × 3 factorial arrangement with main effects of L-carnitine and day of gestation. Gilts were fed a constant feed allowance of 1.75 kg/d and a top-dress containing either 0 or 50 ppm of L-carnitine starting on the first day of breeding through the allotted gestation length (40, 55, or 70). No differences (P > 0.16) were observed for BW or estimated protein or fat mass at any gestation length. Gilts fed L-carnitine tended to have greater (P = 0.10) backfat at d 40 and were numerically heavier at d 70 compared to control gilts. No differences (P > 0.77) were observed in circulating total and free carnitine at breeding, but concentrations increased (P < 0.01) as gestation length increased for the gilts fed L-carnitine compared to those fed the control diet. Fetuses from the gilts fed L-carnitine tended to be heavier (P = 0.06) and fetal circulating IGF-II lower (P = 0.09) at day 70 compared to the fetuses from the control gilts. Insulin-like growth factor-I (IGF-I) mRNA was lower (P = 0.05) in fetal hepatic tissue in fetuses collected from gilts fed supplemental L-carnitine. Insulin-like growth factor binding protein-3 (IGFBP-3; P = 0.05) and IGFBP-5 mRNA increased (P = 0.01) and IGF-I mRNA numerically increased (P = 0.16) in the endometrium of gilts supplemented with L-carnitine. At d 55 or 70 gestation, fetuses were removed for collection of porcine embryonic myoblasts (PEM) from the semitendinosus. There were no treatment differences (P > 0.10) for the expression of IGF-I, IGF-II or IGFBP-5 mRNA levels. However, PEM isolated from fetuses collected from gilts fed L-carnitine had lower (P = 0.08) IGFBP-3 mRNA levels compared to the controls. Myoblasts isolated from fetuses obtained from gilts fed L-carnitine had greater (P = 0.09; 8.8 %) 5.1H11 monoclonal antibody attachment after 72 h in culture. Although not significant (P = 0.20), the total number of PEM in the S phase of the cell cycle was 4.7 % greater in PEM collected from fetuses from gilts fed L-carnitine compared to the controls. This study shows L-carnitine supplementation to gestating gilts has beneficial effects on average fetal weight, due in part to changes in the expression of the IGF axis at the fetal-maternal interface in swine. These changes in the IGF axis play a fundamental role in porcine fetal growth and development due to enhanced proliferation and delayed differentiation of PEM.

This work deals with the determination of L-carnitine in food supplements. L-carnitine is a substance naturally occurring in organism, essential for metabolism of fatty acids. In food supplements is used especially for reducing body weight as a "fat burner". L-carnitine is popular with athletes for improve athletic performance. While data are not available to support these positive effects of carnitine, the positive results of carnitine supplementation in the medicine were found, mainly on cardiovascular system. The literary part of this study describes the properties and the use of carnitine in the diet of human. Furthermore, a review of methods used for determination of L-carnitine in variety of samples is mentioned. In the experimental section the efficiency of SPE method for preparation matrix of real samples was investigated. A cation exchange solid phase extraction seems to be the most effective. The optimal conditions for isolation of L-carnitine by SPE extraction were not developed. L-carnitine was determined in six various food supplements by reverse phase chromatography with UV/VIS detection.

Zusammenfassung: Effekte der L-Carnitinsupplementierung auf das metabolische Profil adipöser und insulinre- sistenter Ponys im Verlaufe einer mehrwöchigen Körpergewichtsreduktion Author: Uta Schmengler Institut für Tierernährung, Ernährungsschäden und Diätetik, Veterinärmedizinische Fakultät, Universität Leipzig Eingereicht im September 2012 76 S., 16 Abb., 23 Tab., 169 Lit., Anhang Einleitung: Das ”Equine Metabolische Syndrom” ist gekennzeichnet durch eine regionale oder generalisierte Adipositas, eine periphere Insulinresistenz sowie akute oder chronische Hufreheschübe. Die Ursache ist in einer bedarfsübersteigenden, hochkalorischen Fütterung und einem relativen Bewegungsmangel zu suchen, wobei auch der genetischen Prädisposition spezieller Rassen eine gewisse Bedeutung zukommt. Ziel dieser Studie war die Untersuchung der Effekte einer L-Carnitinsupplementierung in Kombination mit einer restriktiven Füt- terung und täglicher moderater Bewegung auf Körpermasseverlust, Insulinsensitivität und ausgewählte Parameter des Energiestoffwechsels adipöser und insulinresistenter Ponys. Material und Methoden: Für die placebokontrollierte Doppelblindstudie wurden 16 adipöse Ponys per Losverfahren in zwei Gruppen (N=8) eingeteilt. Zu Versuchsbeginn wiesen die Ponys einen mittleren Body Condition Score von 8,0±2,0 (Skala 1-9) und einen mittleren Cresty Neck Score von 4,0±1,0 (Skala 0-5) auf. Während des 14-wöchigen Körpermassere- duktionsprogramms wurden die Ponys restriktiv gefüttert mit 1 - 1,2 kg Heu/100 kg KM/d. Zusätzlich erhielten 8 Ponys eine L-Carnitin-Zulage (1,3 g/100 kg KM/2d) und 8 Tiere ein Placebo in Form einer Kieselsäureverbindung (1,3 g/100 kg KM/ 2d). Die Ergänzungen wur- den in einem Gemisch aus Grünmehl (50 g/2d) und Mineralfutter verabreicht. Über die 14-wöchige Versuchszeit wurde ein Bewegungsprogramm an sechs Tagen in der Woche durch- geführt, das 25 Minuten Schritt und 15 Minuten Trab beinhaltete. Zu Versuchsbeginn und nach Versuchsende wurde mit beiden Versuchsgruppen ein Frequently sampled intravenous glucose tolerance test (FSIGTT) zur Überprüfung der Insulinsensitivität durchgeführt. Über die gesamte Versuchszeit wurden wöchentlich Blutproben gewonnen zur Bestimmung der ba- salen Serum-Insulinaktivität und Plasma-Glucosekonzentration sowie der Konzentration der Freien Fettsäuren (FFS), Triacylglyceride (TAG), Harnstoff und Betahydroxybutyrat (BHB) im Serum. Die Körpermasseverluste wurden über wöchentliche Wägungen sowie Ermittlung von BCS und CNS kontrolliert. Die statistische Überprüfung wurde anhand parametrischer (ANOVA) und nicht-parametrischer Tests (Wilcoxon signed rank test) durchgeführt, die Kal- kulation der Insulinsensitivität erfolgte über das Minimalmodell anhand eines Computerpro- gramms (MINMOD). Ergebnisse: Im Mittel verloren die Ponys über den Versuchszeitraum von 14 Wochen 1- 3% ihrer Körpermasse pro Woche (Zeit: p < 0, 01, Behandlung: p=0,79), was einem totalen Körpermasseverlust von 14,3±% entsprach. Der BCS reduzierte sich in beiden Versuchs- gruppen um eine Differenz von 3 Einheiten, der CNS verringerte sich in der Carnitingrup- pe (GC ) um eine Differenz von 1,4 und in der Placebogruppe (GP ) um eine Differenz von 1,9 Einheiten. Der Körpermasseverlust war von einer signifikanten Verbesserung der Insu- linsensitivität (Zeit p < 0, 01, Behandlung: p=0,39) begleitet. Die Kalkulation der Insulin- sensitivität im Minimalmodell zeigte eine signifikante Erhöhung der SI-Werte am Versuch- sende in beiden Versuchsgruppen (Beginn Studie GC : 0,76±0,88 l/min/μU*10−4 und GP : 1,61±1,31 l/min/μU*10−4 ; Ende Studie GC : 5,45±0,81 l/min/μU*10−4 und GP : 6,08±2,98 l/min/μU*10−4 ). Signifikante, zeitabhängige Veränderungen wurden auch für die metabo- lischen Parameter beobachtet: Plasma-Glucose und Serum-Insulin reagierten mit einem si- gnifikanten Abfall (Glucose GC : 4,5±0,32 mmol/l vs. 4,21±0,61 mmol/l und Glucose GP : 4,34±0,62 mmol/l vs. 3,86±0,34 mmol/l; Insulin GC : 23,71±32,77 μU/ml vs. 3,67±3,94 μU/ml und GP : 13,55±12,67 μU/ml vs. 1,01±1,09 μU/ml). Dabei kam es zu einem signi- fikanten Anstieg des Serum-Harnstoffs (GC : 3,47±0,73 mmol/l vs. 4,31±1,06 mmol/l und GP : 3,71±0,79 mmol/l vs. 4,9±1,23 mmol/l) sowie der Serum-FFS (GC : 157±95 μmol/l vs. 731±138 μmol/l und GP : 113±63 μmol/l vs. 686±142 μmol/l) und Serum-TAG (GC : 0,53±0,28 mmol/l vs. 0,94±0,61 mmol/l und GP : 0,45±0,23 mmol/l vs. 0,64±0,25 mmol/l). Bezüglich der L-Carnitinsupplementierung wurden keine weiteren Effekte verzeichnet. Schlussfolgerungen: Die restriktive Energiezufuhr von 7 MJ DE/100 kg KM entspre- chend einer Heuzulage von 1 kg/100 kg KM führte zu KM-Verlusten von 1-3 %. Eine Kör- permassereduktion zeigte deutliche Auswirkungen auf den Glucose- und Lipidmetabolismus und führte zu einer signifikanten Verbesserung der Insulinsensitivität, wohingegen die L- Carnitinsupplementierung keine weiteren Effekte auf den Glucosestoffwechsel herbeiführte. Eine bedarfsdeckende Eigensynthese von L-Carnitin ist beim Pony offensichtlich auch im Zu- stand der Insulinresistenz gewährleistet und reicht aus um die obligatorischen Funktionen L-Carnitins im Energiestoffwechsel zu erfüllen. / Summary: The effects of L-carnitine supplementation on body weight losses and metabolic profile in obese and insulin resistant ponies during a several weeks lasting bodyweight reduction pro- gramme Author: Uta Schmengler Institute of Animal Nutrition, Nutrition Diseases and Dietetics, Faculty of Veterinary Medi- cine, University of Leipzig Submitted in September 2012 76 p., 16 fig., 23 tab., 169 ref., appendix Introduction: Insulin resistance, local or general adiposity and the predisposition towards acute or chronical laminitis are components of the equine metabolic syndrome. Contributing factors for this syndrome are the intake and the quality of a high caloric feed by a lack of physical exersice. Howewer, the genetically predisposition of so called ”easy keepers” seems to play a role in pathogenesis. The objective of this study was to investigate the effects of L- carnitine supplementation in combination with a body weight reduction programme (BWRP) on body weight (BW) losses, insulin sensitivity and selected metabolic parameters in obese and insulin resistant ponies. Material und methods: 16 obese ponies (mean BCS = 8.0±2.0, mean CNS = 4.0±1.0) were assigned to a randomized double blind, placebo-controlled study. The ponies werde di- vided into two equal groups (N=8). During a 14 weeks lasting BWRP the ponies were fed 1.0-1.2 kg hay/100 kg BW daily. Additionally, 8 ponies were supplemented with L-carnitine (1.3g/100 kg BW) and 8 ponies were supplemented with a placebo (1.3g/100 kg BW). The supplements were offered in a mixture of 50 g grass meal and 50 g of a commercial mineral mixture, twice a day. During BWRP ponies were exercised a low-intensity protocol 6 days a week (daily 25 min walk and 15 min trot across the countryside). A frequently sampled intravenous glucose tolerance test (FSIGTT) was undertaken in order to assess insulin sen- sitivity at the beginning and the end of the study. Routine blood samples were collected for analysis of plasma glucose, serum insulin, free fatty acids (FFA), triglycerides (TG), urea and beta-hydroxybutyrate (BHB). Ponies were weighed weekly after 12 h of feed restriction by using an electronic scale for large animals. BCS and CNS were recorded weekly by the same 2 observers throughout the study. The statistical analysis was performed by parametric and non-parametric tests (ANOVA and Wilcoxon ranked test). The minimal modell calcu- lation of insulin sensitivity (SI) from FSIGTT was calculated by the computer programme (MINMOD). Results: Ponies lost 1-3% BW per week over the BWRP (time P<0.01, L-carnitine supple- mentation P=0.79), meaning a total body weight loss of 14.3%. BCS decreased in both groups with a difference of three points and CNS was reduced with a difference of 1.4-1.9 points. BW losses were accompanied by a significant improvement in insulin sensitivity (Time: P<0.01, L-carnitine supplementation: P=0.39). The calculation for SI-values by the minimalmodell showed a significant increase in L-carnitine group (GC ) and placebo group (GP ) in the end of the study. (GC : 0.76±0.88 L/min/μU*10−4 to 5.45±0.81 L/min/μU*10−4 , GP : 1.61±1.31 L/min/μU*10−4 to 6.08±2.98 L/min/μU*10−4 ). Significant time related decreases were observed for plasma glucose (GC : 4.5±0.32 mmol/L to 4.21±0.61 mmol/L, GP : 4.34±0.62 mmol/L to 3.86±0.34 mmol/L) and serum insulin (GC : 23.71±32.77 μU/mL to 3.67±3.94 μU/mL, GP : 13.55±12.67 μU/mL to 1.01±1.09 μU/mL). A significant increase was observed for serum urea (GC : 3.47±0.73 mmol/L to 4.31±1.06 mmol/L, GP : 3.71±0.79 mmol/L to 4.9±1.23 mmol/L), FFA (GC : 157±95 μmol/L to 731±138 μmol/L und GP : 113±63 μmol/L to 686±142 μmol/L) and TG (GC : 0.53±0.28 mmol/L to 0.94±0.61 mmol/L, GP : 0.45±0.23 mmol/L to 0.64±0.25 mmol/L) during BWRP. There was no further improvement in metabolic responses by L-carnitine supplementation. Conclusions: Energy intake of 7 MJ DE/100 kg BW leads to bodyweight losses of 1- 3%, herby improving insulin sensitivity and glucose metabolism. L-carnitine supplementation does not further improve glucose or fat metabolism, suggesting that endogenous L-carnitine synthesis was sufficient to facilitate energy metabolism in obese and insulin resistant ponies.

Knowledge on the precise effects of surplus glucose supply in dairy cows is limited by the lack of information on how intermediary metabolism adapts at different levels of glucose availability. Therefore, a gradual increase of glucose supply via intravenous glucose infusion was used in the present study to test the dose effect of surplus provision of glucose on the metabolic status and milk production of dairy cows. Furthermore, the effects of increasing levels of surplus glucose on mRNA expressions and activities of rate-limiting enzymes involved in hepatic gluconeogenesis were investigated. Based on a previous finding that a positive energy balance may decrease hepatic carnitine palmitoyltransferase (CPT) enzyme activity, it was also of interest whether skeletal muscle CPT activity is downregulated in a similar manner during positive energy balance. Twelve midlactating Holstein-Friesian dairy cows were continuously infused over a 28-d experimental period with either saline (SI group, six cows) or 40% glucose solutions (GI group, six cows). The infusion dose was calculated as a percentage of the daily energy (NEL) requirements by the animal, starting at 0% on d 0 and increasing gradually by 1.25%/d until a maximum dose of 30% was reached by d 24. Dose was then maintained at 30% NEL requirement for 5 d. No infusions were made between d 29-32. Liver and skeletal muscle biopsies were taken on d 0, 8, 16, 24, and 32. Body weight (BW) and back fat thickness (BFT) were recorded on biopsies days. Blood samples were taken every 2 d. In addition, blood samples over 24 h (6-h intervals) were taken the days before each biopsy. Milk and urine samples were taken on biopsies days. BW and BFT increased linearly with increasing glucose dose for GI cows. No differences were observed in the dry matter intake, milk energy output, and energy corrected milk yield between groups. However, milk protein percentage and yield increased linearly in the GI group. Only occasional increases in blood glucose and insulin concentrations were observed in blood samples taken at 1000 h every 2 d. However, during infusion dose of 30% NEL requirements on d 24, GI cows developed postprandial hyperglycemia associated with hyperinsulinemia, coinciding with glucosuria. The revised quantitative insulin sensitivity check index (RQUIKI) indicated linear development of insulin resistance for the GI treatment. GI decreased serum concentrations of beta-hydroxybutyrate (BHBA) and blood urea nitrogen and tended to decrease the serum concentration of non-esterified fatty acids (NEFA). Liver glycogen content increased, while glycogen content in skeletal muscle only tended to increase by GI. No significant changes were observed in the activities and relative mRNA expression levels of hepatic phosphoenolpyruvate carboxykinase and glucose 6-phospatase. The activity of fructose 1,6-bisphosphatase (FBPase) and relative mRNA expression levels of pyruvate carboxylase (PC) were decreased in the GI group but only during the high dose of glucose infusion. Hepatic CPT activity decreased with GI and remained decreased on d 32. The hepatic expression levels of CPT-1A and CPT-2 mRNA were not significantly altered but tended to reflect the changes in enzyme activity. No effect of glucose infusion was observed on skeletal muscle CPT activity. The aforementioned adaptations were reversed four days after the end of glucose infusions except for those of BW, BFT, and lipid metabolism (i.e. serum BHBA and NEFA concentrations, hepatic CPT activity). It is concluded that mid-lactation dairy cows on an energy-balanced diet direct intravenously infused glucose predominantly to body fat reserves but not to increased lactation performance. Cows rapidly adapted to increasing glucose supply but experienced dose-dependent development of insulin resistance corresponding with postprandial hyperglycemia/hyperinsulinemia and glucosuria at dosages equivalent to 30% NEL requirements. The catalytic capacity of key hepatic gluconeogenesis enzymes in mid-lactating dairy cows is not significantly affected by nutritionally relevant increases of glucose supply. Only very high dosages selectively suppress PC transcription and FBPase activity. Finally, it can be concluded that suppression of CPT activity by positive energy balance appears to be specific for the liver in midlactating dairy cows.

No description available.

La forme canadienne-française du syndrome de Leigh (LSFC) est une maladie métabolique associée à une déficience en cytochrome oxydase (COX) et caractérisée par des crises d’acidose lactique, menant à une mort prématurée. Les mécanismes qui sous-tendent l’induction des crises restent inconnus et il n’existe aucune thérapie efficace pour les prévenir. Cette étude vise à caractériser l'effet de facteurs métaboliques périphériques potentiellement altérés chez les patients LSFC sur la mort de lignées cellulaires issues de ces patients et de témoins puis, à identifier des agents thérapeutiques pouvant la prévenir. Nous postulons que (i) ces facteurs métaboliques induiront une mort prématurée des cellules de patients et que (ii) les interventions susceptibles de la prévenir pallieront les conséquences de la déficience en COX, soit la diminution des taux d’adénosine triphosphate (ATP) et l’augmentation du stress oxydant, du nicotinamide adénine dinucléotide (NADH) et des lipides toxiques. Un criblage de 8 facteurs sanguins et 10 agents thérapeutiques a été réalisé. Les paramètres mesurés incluent la nécrose, l’apoptose, l’ATP et l’activité de la COX. Les fibroblastes LSFC sont plus susceptibles à la mort par nécrose (39±6%) induite par du palmitate plus lactate, un effet associé à des niveaux d’ATP diminués (53±8%). La mort cellulaire est réduite de moitié par l’ajout combiné d’agents ciblant le NADH, l’ATP et les lipides toxiques, alors que l’ajout d’antioxydants l’augmente. Ainsi, un excès de nutriments pourrait induire la mort prématurée des cellules LSFC et, pour atténuer cette mort, il serait important de combiner plusieurs interventions ciblant différents mécanismes. / Leigh syndrome French-Canadian variant (LSFC) is a metabolic disease associated with cytochrome c oxidase (COX) deficiency and characterized by episodes of lactic acidosis, referred to as “crisis”, leading to death at an early age. The mechanisms underlying a crisis and its cellular consequences remain elusive, and there is no effective therapy. The aim of this study was to characterize the effect of peripheral metabolic factors that are potentially altered in patients with LSFC on their cells death and to identify therapeutic agents able to prevent them using cell-lineage from LSFC patients and controls. The hypothesis are that (i) these metabolic factors can induce premature death in patient cells, and (ii) interventions that could rescue these cells may target potential consequences of COX deficiency, namely low adenosine triphosphate (ATP), high nicotinamide adenine dinucleotide (NADH) and toxic lipids, as well as oxidative stress. A screening of 8 blood factors and 10 therapeutic agents was conducted in fibroblasts. Parameter measured included cell death by necrosis and apoptosis, as well as ATP level and COX activity. LSFC fibroblasts were more susceptible to necrosis (39±6%) induced by high palmitate plus lactate and this was associated with a lower ATP (53±8%). Cell death decreased 2-fold with combined interventions, which presumably act on NADH, ATP, and the accumulation of toxic lipids, but increased with antioxidants. Collectively, our results emphasize the importance of nutrient overload as a factor eliciting premature cell death in LSFC cells and of combining interventions acting through various mechanisms for cell death rescue.

La forme canadienne-française du syndrome de Leigh (LSFC) est une maladie métabolique associée à une déficience en cytochrome oxydase (COX) et caractérisée par des crises d’acidose lactique, menant à une mort prématurée. Les mécanismes qui sous-tendent l’induction des crises restent inconnus et il n’existe aucune thérapie efficace pour les prévenir. Cette étude vise à caractériser l'effet de facteurs métaboliques périphériques potentiellement altérés chez les patients LSFC sur la mort de lignées cellulaires issues de ces patients et de témoins puis, à identifier des agents thérapeutiques pouvant la prévenir. Nous postulons que (i) ces facteurs métaboliques induiront une mort prématurée des cellules de patients et que (ii) les interventions susceptibles de la prévenir pallieront les conséquences de la déficience en COX, soit la diminution des taux d’adénosine triphosphate (ATP) et l’augmentation du stress oxydant, du nicotinamide adénine dinucléotide (NADH) et des lipides toxiques. Un criblage de 8 facteurs sanguins et 10 agents thérapeutiques a été réalisé. Les paramètres mesurés incluent la nécrose, l’apoptose, l’ATP et l’activité de la COX. Les fibroblastes LSFC sont plus susceptibles à la mort par nécrose (39±6%) induite par du palmitate plus lactate, un effet associé à des niveaux d’ATP diminués (53±8%). La mort cellulaire est réduite de moitié par l’ajout combiné d’agents ciblant le NADH, l’ATP et les lipides toxiques, alors que l’ajout d’antioxydants l’augmente. Ainsi, un excès de nutriments pourrait induire la mort prématurée des cellules LSFC et, pour atténuer cette mort, il serait important de combiner plusieurs interventions ciblant différents mécanismes. / Leigh syndrome French-Canadian variant (LSFC) is a metabolic disease associated with cytochrome c oxidase (COX) deficiency and characterized by episodes of lactic acidosis, referred to as “crisis”, leading to death at an early age. The mechanisms underlying a crisis and its cellular consequences remain elusive, and there is no effective therapy. The aim of this study was to characterize the effect of peripheral metabolic factors that are potentially altered in patients with LSFC on their cells death and to identify therapeutic agents able to prevent them using cell-lineage from LSFC patients and controls. The hypothesis are that (i) these metabolic factors can induce premature death in patient cells, and (ii) interventions that could rescue these cells may target potential consequences of COX deficiency, namely low adenosine triphosphate (ATP), high nicotinamide adenine dinucleotide (NADH) and toxic lipids, as well as oxidative stress. A screening of 8 blood factors and 10 therapeutic agents was conducted in fibroblasts. Parameter measured included cell death by necrosis and apoptosis, as well as ATP level and COX activity. LSFC fibroblasts were more susceptible to necrosis (39±6%) induced by high palmitate plus lactate and this was associated with a lower ATP (53±8%). Cell death decreased 2-fold with combined interventions, which presumably act on NADH, ATP, and the accumulation of toxic lipids, but increased with antioxidants. Collectively, our results emphasize the importance of nutrient overload as a factor eliciting premature cell death in LSFC cells and of combining interventions acting through various mechanisms for cell death rescue.

No presente estudo avaliamos o impacto da adição de fluido folicular (FF) de mulheres inférteis sem e com endometriose em estágios iniciais (I/II) e avançados [(III/IV) sem e com endometrioma] ao meio de maturação in vitro (MIV) sobre as taxas de normalidade meiótica de oócitos bovinos. Avaliamos se a L-carnitina (LC) e os ácidos graxos ômega-3 [n3, ácidos docosahexaenóico (DHA) e eicosapentaenoico (EPA)] são capazes de prevenir os danos meióticos em oócitos bovinos induzidos por FF de mulheres inférteis com endometriose I/II e III/IV durante a MIV. Para isso, realizamos um estudo experimental utilizando modelo bovino. Trinta e duas amostras de FF foram colhidas de 24 mulheres inférteis com endometriose (8 com I/II, 8 com III/IV sem endometrioma e 8 III/IV com endometrioma no ciclo) e 8 sem endometriose (controle) que foram submetidas à estimulação ovariana controlada para realização de injeção intracitoplasmática de espermatozoide. Complexos cumulus-oócitos (CCOs) imaturos de bovinos foram submetidos à MIV divididos em 9 grupos: sem FF (sem-FF), com 1% de FF de mulheres inférteis sem endometriose (FFControle) e com endometriose (FFEI/II, FFEIII/IV e FFEendometrioma) suplementados ou não com LC (0,6mg/mL) e ácidos graxos ômega-3 (0,4 nM de DHA e 0,6 nM de EPA) (FFControle+LC+n3, FFEI/II+LC+n3, FFEIII/IV+LC+n3 e FFEendometrioma+LC+n3). Após 22-24h de MIV, os oócitos foram denudados, fixados e armazenados para realização de imunofluorescência para visualização do fuso meiótico e cromossomos por microscopia confocal. As taxas de metáfase II (MII) e de MII normais foram comparadas entre os 9 grupos utilizando o teste do qui-quadrado (p<0,05). Um total de 1686 CCOs imaturos foram submetidos à MIV, e 1401 oócitos foram visualizados por microscopia confocal. A adição de FF de mulheres com endometriose ao meio de MIV reduziu a taxa de MII normais (FFEI/II: 62,2%, FFEIII/IV: 70,2% e FFEendometrioma: 72,7%) comparado aos grupos sem-FF (87,2%) e FFControle (87,2%). O grupo FFEendometrioma (69,3%) apresentou a menor taxa de MII comparado a todos os demais grupos (sem-FF: 91,9%, FFControle: 89,2%, FFControle+LC+n3: 89,2%, FFEI/II: 85,4%, FFEI/II+LC+n3: 85,3%, FFEIII/IV: 80,7%, FFEIII/IV+LC+n3: 90,8%, FFEndometrioma+LC+n3: 86,4%). O grupo FFEIII/IV apresentou menor taxa de MII comparado ao grupo sem-FF. No grupo com FFControle, a adição de LC+n3 não alterou as taxas de MII (89,2% vs 89,2) e de MII normais (87,2% vs 82,5%). No grupo FFEI/II, a adição de LC+n3 aumentou a taxa de MII normais (84,5% vs. 62,2%). No grupo FFEIII/IV a adição de LC+n3 aumentou a taxa de MII normais (70,2% vs 84,1%) e de MII (90,8%), que passou a ser semelhante a dos grupos sem-FF e FFControle. No grupo FFEendometrioma a adição de LC+n3 aumentou a taxa de MII normais (86,4%), comparado ao grupo FFEendometrioma (69,3%), a qual foi similar a dos grupos sem-FF e FFControle. Portanto, o FF de mulheres com endometriose prejudica o fuso meiótico e o alinhamento cromossômico de oócitos bovinos, independentemente, do estágio da doença. Entretanto, o avanço da endometriose e a presença de endometrioma parecem ter um impacto ainda mais negativo na qualidade oocitária, prejudicando também a maturação nuclear. A adição de LC+n3 previne os danos meióticos oocitários provocados pelo FF de mulheres com endometriose em estágios iniciais e avançados. Dessa forma, sugerimos que inflamação, o estresse oxidativo e a desregulação da ?-oxidação são fatores envolvidos na alteração da qualidade oocitária e, consequente, piora da fertilidade natural de mulheres com endometriose. / In the present study, we evaluated the impact of the addition of follicular fluid (FF) from infertile women without and with endometriosis in the early (I/II) and advanced stages [(III/IV) with and without endometrioma] to the in vitro maturation (IVM) medium on the meiotic normality rates of bovine oocytes. We evaluated whether L-carnitine (LC) and omega-3 fatty acids [n3, docosahexaenoic (DHA) and eicosapentaenoic acid (EPA)] were able to prevent bovine meiotic oocyte damage induced by FF from infertile women with endometriosis in stages I/II and III/IV during IVM. For this, we performed an experimental study using bovine model. Thirty-two FF samples were collected from 24 infertile women with endometriosis (8 with I/II, 8 with III/IV without endometrioma and 8 III/IV with endometrioma in the cycle) and 8 without endometriosis (control) who underwent to controlled ovarian stimulation for intracytoplasmic sperm injection. Immature cumulus oocytes complexes(COCs) of bovines were submitted to IVM divided into 9 groups: without FF (No-FF), with 1% FF of infertile women without endometriosis (FFControl) and with endometriosis (FFEI/II, FFEIII/IV and FFEendometrioma) supplemented or not with LC (0.6 mg/mL) and omega-3 fatty acids (0.4 nM DHA and 0.6 nM EPA) (FFControl+LC+n3, FFEI/II+LC+n3, FFEIII/IV+LC+n3 and FFEendometrioma+LC+n3). After 22-24h of IVM, the oocytes were denuded, fixed and stored for subsequent immunofluorescence to visualize the meiotic spindle and chromosomes by confocal microscopy. The metaphase II (MII) and normal MII rates were compared between the 9 groups using the chi-square test (p <0.05). A total of 1686 immature COCs were submitted to IVM, and 1401 oocytes were visualized by confocal microscopy. Addition of FF from women with endometriosis to the IVM medium decreased the rate of normal MII (FFEI/II: 62.2%, FFEIII/IV: 70.2% and FFEendometrioma: 72.7%) compared to the No-FF (87.2%) and FFControl (87.2%) groups. The FFEendometrioma group (69.3%) presented the lowest rate of MII compared to all other groups (No-FF: 91.9%, FFControl: 89.2%, FFControl+LC+n3: 89.2%, FFEI/II: 85.4%, FFEI/II+LC+n3: 85.3%, FFEIII/IV: 80.7%, FFEIII/IV+LC+n3: 90.8%, FFEndometrioma+LC+n3: 86.4%). The FFEIII/IV group had a lower MII rate compared to the No-FF group. In the group with FFControl, the addition of LC+n3 did not change the rates of MII (89.2% vs 89.2%) and normal MII (87.2% vs 82.5%). In the FFEI/II group, the addition of LC+n3 increased the normal MII rate (84.5% vs 62.2%). In the FFEIII/IV group, the addition of LC+n3 increased the normal MII rate (70.2% vs 84.1%) and MII (90.8%), which was similar to that of the No-FF and FFControl. In the FFEendometrioma group, the addition of LC+n3 increased the normal MII rate (69.3% vs 86.4%) which was similar to No-FF and FFControl groups. Therefore, the FF of women with endometriosis impairs the meiotic spindle and the chromosomal alignment of bovine oocytes, regardless of the stage of the disease. However, the progression of endometriosis and the presence of endometrioma appear to have an even more negative impact on oocyte quality, and also impairs nuclear maturation. The addition of LC+n3 prevents meiotic oocyte damages induced by FF from women with endometriosis in the early and advanced stages. Thus, we suggest that inflammation, oxidative stress and deregulation of ?-oxidation are factors involved in the alteration of oocyte quality and, consequently, worsening of the natural fertility of women with endometriosis.

O uso de suplementos alimentares à base de carnitina tem se tornado bastante popular dentre atletas. Nos seus possíveis efeitos biológicos, constam o emagrecimento e o melhor condicionamento aeróbio frente ao exercício físico. Embora o uso difundido também entre não-atletas, há poucas evidências científicas nestes grupos populacionais, particularmente adultos. O objetivo deste estudo foi avaliar os efeitos da suplementação de L-carnitina associada ao exercício físico aeróbio sobre a composição corporal e lipídica sanguínea, gasto energético de repouso e desempenho aeróbio de adultos clinicamente saudáveis. Foram selecionados 21 indivíduos voluntários de 40 a 58 anos de idade, de ambos os sexos (9 homens e 12 mulheres), com índice de Massa Corporal (IMC) entre 25 e 35 kg/m2, participantes de protocolo de exercícios físicos aeróbios supervisionados (80min/sessão, 3-5x/semana, 70 a 80% da freqüência cardíaca máxima para idade) há pelo menos 12 semanas. Após avaliação inicial (M0), foram divididos aleatoriamente em grupos: suplementado (G1; N=11), recebeu 1,8g/dia de L-carnitina e placebo (G2; N=10), recebeu maltodextrina, ambos mantidos nesta intervenção dietética por 30 dias consecutivos. Concluído o período dietético (M1), foram repetidas as avaliações de M0, nas situações de repouso (peso, estatura para cálculo do IMC, circunferência de abdômen, % de gordura, gasto energético de repouso, ingestão alimentar, colesterol e frações e triglicerídios) e esforço físico em esteira ergométrica (VO2máx, limiar anaeróbio, quociente respiratório e variação dos ácidos graxos livres plasmáticos). Houve ligeiro aumento do V02máx e limiar anaeróbio em ambos os grupos e reclassificação do LDL-c no grupo placebo. Os demais valores de ingestão alimentar, composição corporal, lipidemia e gasto energético não sofreram influência significativa do período de exercício ou tratamento dietético. As concentrações de ácidos graxos livres aumentaram durante o esforço físico em esteira, mas sem significância. Conclui-se que o efeito adicional da suplementação de L-carnitina em adultos exercitados regularmente é mínimo nas variações da composição corporal e sanguínea, no gasto energético, uso de substratos energéticos e no condicionamento aeróbio. / The use of nutritional supplements such as carnitine has been widely spread over among athletes. The refered advantages are related to possible weight loss and cardiorespiratory fitness. However, besides widely used in active people (non athletes) there has been little scientific based evidences in this group, specifically in adults. The purpose of the study was to investigate the additional effects of L-carnitine supplemented to exercised subjects on their body composition, blood lipid profile, resting metabolic rate and aerobic performance. Twenty-one volunteers (9 males and 12 females), 40 to 58 years old, body mass index (BMI) values between 25 and 35 kg/m2, were engaged in aerobic exercise program (80 min/session, 3-5 days/week, 70 a 80% of maximum heart rate-HRmáx) at least 12 weeks. After the first test (M0) the subjects were randomly assigned in two groups: L-carnitine (G1; N=11), receiving orally L-carnitine (1,8g/day) or placebo (G2; N=10), receiving maltodextrine during 30 consecutive days. After the dietary intervention (M 1), the assessment tests were repeated in both, resting (body mass, height, BMI calculation, resting energy expenditure, dietary intake, body fat and lipid profile) and exercised condition in a treadmill (VO2max, anaerobic threshold, respiratory exchange ratio and the variation on free fatty acids levels). VO2max and anaerobic threshold were increased in both groups and LDL-c downgraded in the placebo group. No significant changes were found due to either training or dietary supplementation in dietary intake, body composition, lipid profile and energy expenditure. Plasma free fatty acids levels increased, but not significantly, during the 30 min treadmill exercise. Thus, the additional effects of L-carnitine supplementation in moderate active adults were not enough to promote significant changes in body composition, lipid profile, energy expenditure, substrate utilization and aerobic fitness.