Tang, Leung Sang Nelson. / "February 2002." / Thesis (M.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 185-206). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Decline in cardiac pump function is a hallmark of aging where mitochondrial decay is an important underlying cause. Although certainly multifactorial in nature, both dysfunction of the machinery involved in the chemiosmotic process of energy transduction and lower capacity to maintain fatty acid-driven respiration are identified as intrinsic factors of mitochondrial decay in the aged myocardium. Age-associated destabilization of electron transport supercomplexes as a potential factor of mitochondrial decay in the rat heart. Defective operation of the electron transport chain (ETC) constitutes a key mechanism involved in the age-associated loss of mitochondrial energy metabolism. Nevertheless, the molecular events underlying inefficient electron flux that ultimately leads to higher superoxide appearance and impaired respiration are not fully known. As recent biophysical evidence shows that the ETC may form large macromolecular assemblies (i.e. supercomplexes) that disintegrate in certain pathologies (e.g. heart failure or Barth syndrome) reminiscent of aging, we investigated the hypothesis that alterations in supercomplexes are partly responsible for the age-related loss of cardiac ETC function. In this dissertation, age-associated changes in supercomplex organization and stability were investigated in subsarcolemmal (SSM) and interfibrillary (IFM) mitochondria isolated from cardiac tissue from young (3-5 months) and old (24-28 months) male Fischer 344 rats. Blue native-PAGE (BN-PAGE) analysis of digitonin-solubilized mitochondrial membranes coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to investigate supercomplex organization. Results show that both SSM and IFM display supercomplexes comprised of various stoichiometries of complexes I, III and IV (never complex II), which typically organize as high mass (1500-2300 kDa) assemblies containing up to four copies of complex IV (i.e. I���III���IV[subscript N]-type supercomplexes). Interestingly, analysis of IFM proteins showed that, in general, supercomplex levels declined by up to 15 % (p < 0.05) with age; however, different degrees of supercomplex deterioration were observed, depending on the particular supercomplex investigated. Supercomplexes of the highest molecular weights (i.e. 1900-2300 kDa), which were also composed of the most complex stoichiometries (i.e. I1III2IVN, N ��� 2), were primarily lost with age. In particular, I���III���IV���, I���III���IV��� and I���III���IV��� supercomplexes were found to decline by 13% (p < 0.05), 30% (p < 0.05) and 45% (p < 0.05), respectively, on an age basis. Therefore, the age-associated loss of supercomplexes in IFM stems from destabilization of the assemblies that comprise several copies of complex IV, which could partially limit proper electron transfer to O��� for its reduction, affecting mitochondrial respiratory capacity. In contrast to IFM, the aging defects of SSM supercomplexes appeared to be confined to the assembly comprised of only one copy of complex IV (I���III���IV���, 1700 kDa) (37% loss; p = 0.06), while the higher molecular weight supercomplex sub-types that were most affected in IFM (i.e. I���III���IV[subscript N], N ��� 2) were not significantly altered with age. Thus, the results from this dissertation indicate that mitochondria from different subcellular locations in the myocyte show different degrees of supercomplex destabilization in the aging rat heart. The more robust supercomplex deficits noted for IFM fit well with previous observations that electron transport characteristics of this subpopulation are more adversely affected with age than SSM. Although the underlying factor(s) of supercomplex deterioration are not fully known, the hypothesis that age-related alterations of certain constituents of the IMM (e.g. cardiolipin) may be important factors of supercomplex destabilization in cardiac mitochondria was investigated in this dissertation. To this end, LC-MS/MS characterization of supercomplex proteins and HPLC analysis of cardiolipin were used as approaches to elucidate potential factor(s) of supercomplex destabilization in the aging rat heart. Age-related alterations of cardiolipin levels and its acyl-chain content showed a strong parallel to the age-associated destabilization of supercomplexes. Specifically, cardiolipin levels declined by 10% (p < 0.05) in IFM, the mitochondrial subpopulation displaying the highest degree of supercomplex deterioration. In addition, the content of (18:2)���-cardiolipin, the predominant species in the heart, was found to decline by 50% (p < 0.05) on average in both populations of cardiac mitochondria. Therefore, the data presented in this dissertation indicate that changes in cardiolipin may be at least one of the factors involved in supercomplex destabilization in the aging heart. Age-related decline in carnitine palmitoyltransferase I (CPT1) activity as a mitochondrial lesion that limits fatty acid catabolism in the rat heart. Loss of fatty acid utilization, another intrinsic factor of mitochondrial decay in the aged myocardium, has been associated with age-related alterations in the activity of carnitine palmitoyltransferase 1 (CPT1), the rate-controlling enzyme for overall fatty acid ��-oxidation. Nevertheless, the exact molecular mechanism involved in the age-related loss of fatty acid-driven bioenergetics is not fully understood. In this dissertation, it was also investigated whether the aging lesion for fatty oxidation lies in a particular mitochondrial subpopulation or more generally results from cardiac decrements in L-carnitine levels. In order to clarify the role of each one of these factors, the effect of long-term dietary supplementation with the L-carnitine analogue, acetyl-L-carnitine (ALCAR), was also investigated. Results show that aging selectively decreases CPT1 activity in IFM by reducing enzyme catalytic efficiency for palmitoyl-CoA. IFM displayed a 28% (p < 0.05) loss of CPT1 activity, which correlated with a decline (41%, p < 0.05) in palmitoyl-CoA-driven state 3 respiration. Interestingly, SSM had preserved enzyme function and efficiently utilized palmitate. Analysis of IFM CPT1 kinetics showed both diminished V[subscript max] and K[subscript m] (60% and 49% respectively, p < 0.05) when palmitoyl-CoA was the substrate. However, no age-related changes in enzyme kinetics were evident with respect to L-carnitine. ALCAR supplementation restored CPT1 activity in heart IFM, but not apparently through remediation of L-carnitine levels. Rather, ALCAR influenced enzyme activity over time, potentially by modulating conditions in the aging heart that ultimately affect palmitoyl-CoA binding and CPT1 kinetics. In conclusion, this dissertation presents a characterization of age-associated alterations in the macromolecular organization of the IMM components that could partly explain the loss of mitochondrial oxidative capacity that affects the aging heart. In addition, the characterization of an age-related lesion of the controlling enzyme for ��-oxidation is presented as another important factor that limits mitochondrial function and energy metabolism in cardiac mitochondria. / Graduation date: 2013

Plusieurs maladies entraînent soit une augmentation soit une diminution du taux des acides gras (AG) et de leurs dérivés circulants, notamment les acyl-carnitines (AC). Ce changement a été soupçonné comme étant la cause de importants dérangements électriques. Nous avons montré que les AC à chaine longue (LCAC) du côté extracellulaire modulent le canal hERG de façon spécifique, modulant sa amplitude de courant et sa cinétique. Aucun AC testé n’a eu d’effet en intracellulaire. La CAR et les MCAC n’ont eu aucun effet. Les AC ne modulent pas les courants IKS et IK1. Le canal Cav1.2 est modulé par C16-CAR et le C16 dans la lignée HEK293-ICaL et dans des cardiomyocytes de rat. En condition physiologique il existe donc un lien strict entre le métabolisme énergétique et activité électrique cardiaque qui entraine une modulation permanente du canal hERG par les LCAC. La régulation par les LCAC du canal hERG et peut être celle du canal ICaL, pourraient participer au dérangement électrique à l’origine du déclenchement de troubles du rythme cardiaque retrouvé dans certaines maladies. / Several diseases can cause either an increase or a decrease in the rate of fatty acids (FAs) and their derivatives circulating, including acyl-carnitines (AC). This change is suspected as being the cause of major cardiac electrical perturbations. We have shown that long-chain AC (LCAC) modulate specifically by the extracellular side the hERG channel, regulating its current amplitude and kinetics. All AC tested had no effect when applied intracellularly. Carnitine and medium chain AC had no effect on hERG. LCAC does not modulate IK1 and IKS. Cav1.2 channel is modulated by C16 and C16-CAR in line HEK293-ICaL and rat cardiomyocytes. In physiological conditions there exists a strict link between energy metabolism and cardiac electrical activity which causes a permanent modulation of hERG channel by the LCAC. Regulation by the LCAC of the hERG channel and maybe ICaL, could participate in the electrical disturbance causing the onset of cardiac arrhythmia found in certain diseases.

Doctor of Philosophy / Department of Animal Sciences and Industry / Michael D. Tokach / Four experiments were conducted to determine the interactive effects of dietary L-carnitine and ractopamine HCl (ractopamine) on finishing pig growth performance. In analysis of treatments common to all experiments, ractopamine increased (P < 0.01) ADG and G:F compared to pigs not fed ractopamine. Added L-carnitine tended to increase (P < 0.07) ADG and improved (P < 0.01) G:F compared to pigs not fed L-carnitine. Three experiments were conducted to determine the effects of L-carnitine and ractopamine on carcass characteristics and meat quality. In Exp. 1, drip loss decreased (linear, P < 0.04) in pigs fed increasing L-carnitine. In Exp. 2, drip loss decreased (P < 0.04) with increasing L-carnitine when fed with ractopamine. Percentage lean was higher (P < 0.01) for pigs fed ractopamine. In Exp. 3, lean percentage increased (P < 0.03) in pigs fed L-carnitine or ractopamine. Pigs fed L-carnitine tended (P < 0.06) to have decreased drip loss. These results suggest that ractopamine increases carcass leanness and L-carnitine reduces drip loss when fed in combination with ractopamine. Two experiments were conducted to determine the effects of L-carnitine and ractopamine on the metabolic response to handling. Non-gentle handling increased (P < 0.01) lactate and rectal temperature, and decreased pH. In Exp. 1, non-gentle handled pigs fed ractopamine had decreased (P < 0.01) pH and increased temperature and tended (P < 0.09) to have higher lactate than other pigs. In Exp. 2, lactate and temperature changes from immediately post-handling to 1 h post-handling were not different for pigs fed L-carnitine or ractopamine suggesting that L-carnitine did not decrease recovery time of pigs subjected to non-gentle handling or fed ractopamine. These results suggest that pigs fed ractopamine are more susceptible to stress when handled aggressively. Because carnitine did not alleviate the negative effects of handling for pigs fed ractopamine, the improvement in drip loss from feeding carnitine must be due to a different mode of action.

Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Carnitine plays an essential role in eukaryotic metabolism by mediating the shuttling of activated acyl residues between intracellular compartments. This function of carnitine, referred to as the carnitine shuttle, is supported by the activities of carnitine acyltransferases and carnitine/acylcarnitine transporters, and is reasonably well studied and understood. While this function remains the only metabolically well established role of carnitine, several studies have been reporting beneficial effects associated with dietary carnitine supplementation, and some of those beneficial impacts appear not to be directly linked to shuttle activity. This study makes use of the yeast Saccharomyces cerevisiae as a cellular model system in order to study the impact of carnitine and of the carnitine shuttle on cellular physiology, and also investigates the eukaryotic carnitine biosynthesis pathway. The carnitine shuttle of S. cerevisiae relies on the activity of three carnitine acetyltransferases (CATs), namely Cat2p (located in the peroxisome and mitochondria), Yat1p (on the outer mitochondrial membrane) and Yat2p (in the cytosol), which catalyze the reversible transfer of activated acetyl units between CoA and carnitine. The acetylcarnitine moieties can be transferred across the intracellular membranes of the peroxisomes and mitochondria by the activity of the carnitine/acetylcarnitine translocases. The activated acetyl groups can be transferred back to free CoA-SH and further metabolised. In addition to the carnitine shuttle, yeast can also utilize the glyoxylate cycle for further metabolisation of in particular peroxisomally generated acetyl-CoA. This cycle results in the net production of succinate from two molecules of acetyl-CoA. This dicarboxylic acid can then enter the mitochondria for further metabolism. Partial disruption of the glyoxylate cycle, by deletion of the citrate synthase 2 (CIT2) gene, generates a yeast strain that is completely dependent on the activity of the carnitine shuttle and, as a consequence, on carnitine supplementation for growth on fatty acids and other non-fermentable carbon sources. In this study, we show that all three CATs are required for the function of the carnitine shuttle. Furthermore, overexpression of any of the three enzymes is unable to crosscomplement deletion of any one of the remaining two, suggesting a highly specific role for each CAT in the function of the shuttle. In addition, a role for carnitine that is independent of the carnitine shuttle is described. The data show that carnitine can influence the cellular response to oxidative stresses. Interestingly, carnitine supplementation has a protective effect against certain ROS generating oxidants, but detrimentally impacts cellular survival when combined with thiol modifying agents. Although carnitine is shown to behave like an antioxidant within a cellular context, the molecule is unable to scavenge free radicals. The protective and detrimental impacts are dependent on the general regulators of the cells protection against oxidative stress such as Yap1p and Skn7p. Furthermore, from the results of a microarray based screen, a role for the cytochrome c heme lyase (Cyc3p) in both the protective and detrimental effects of carnitine is described. The requirement of cytochrome c is suggestive of an involvement in apoptotic processes, a hypothesis that is supported by the analysis of the impact of carnitine on genome wide transcription levels. A separate aim of this project involved the cloning and expression in S. cerevisiae of the four genes encoding the enzymes from the eukaryotic carnitine biosynthesis pathway. The cloned genes, expressed from the constitutive PGK1 promoter, were sequentially integrated into the yeast genome, thereby reconstituting the pathway. The results of a plate based screen for carnitine production indicate that the engineered laboratory strains of S. cerevisiae are able to convert trimethyllysine to L-carnitine. This work forms the basis for a larger study that aims to generate carnitine producing industrial yeast strains, which could be used in commercial applications. / AFRIKAANSE OPSOMMING: Karnitien vervul ‘n noodsaaklike rol in eukariotiese metabolisme deur die pendel van asiel residue tussen intersellulêre kompartemente te medieer. Hierdie funksie van karnitien heet “die karnitien-pendel“ en word ondersteun deur verskeie karnitien asieltransferases en karnitine/asielkarnitien oordragsprotiëne. Die rol van die karnitien-pendel is redelik goed gekarakteriseer en is tot op hede die enigste bevestigde rol van karnitien in eukariotiese metabolisme. Verskeie onlangse studies dui egter op voordele geasosieer met karnitien aanvulling, wat in sommige gevalle blyk om onafhanklik te wees van die pendel aktiwiteit van karnitien. Hierdie studie maak gebruik van die gis, Saccharomyces cerevisiae, as ‘n sellulêre model sisteem om die impak van karnitien op sel fisiologie asook die eukariotiese karnitien biosintese pad te bestudeer. Die karnitien-pendel van S. Cerevisiae is afhanklik van die aktiwiteite van drie afsonderlike karnitien asetieltransferases (CATs), naamlik Cat2p (gelokaliseer in die peroksisoom en die mitochondria), Yat1p (op die buitenste membraan van die mitochondria) en Yat2p (in die sitosol). Die drie ensieme kataliseer die omkeerbare oordrag van asetielgroepe tussen CoA en karnitien. Die terugwaartse reaksie stel CoA-SH vry om sodoende verbruik te word in verdere metaboliese reaksies. Gis is in staat om, afsonderlik van die karnitien-pendel, gebruik te maak van die glioksilaat siklus vir verdere metabolisme van asetiel-CoA wat gevorm word in die peroksisoom. Gedeeltelike onderbreking van hierdie siklus deur uitwissing van die sitraat sintase (CIT2) geen, genereer ’n gisras wat afhanklik is van die funksie van die karnitienpendel en ook van karnitien aanvulling vir groei op vetsure en nie-fermenteerbare koolstofbronne. Hierdie studie dui daarop dat al drie CATs noodsaaklik is vir die funksionering van die karnitien-pendel. Ooruitdrukking van enige van die drie ensieme lei slegs tot selfkomplementasie en nie tot kruis-komplementasie van die ander twee CATs nie. Hieruit word ’n hoogs spesifieke rol vir elk van die drie ensieme afgelei. ’n Pendel-onafhanklike rol vir karnitien word ook in hierdie werk uitgewys in die bevordering van weerstand teen oksidatiewe stres. Dit is noemenswaardig dat karnitien ’n beskermende effek het in kombinasie met oksidante wat ROS genereer en ’n nadelige effek in kombinasie met sulfhidriel modifiserende agente. Dit word aangedui dat karnitien antioksidant funksie naboots in die konteks van ’n gis sel terwyl die molekuul nie in staat is om vry radikale te deaktiveer nie. Beide die beskermende asook die nadelige inwerking van karnitien is afhanklik van Yap1p en Skn7p, wat reguleerders is in die algemene beskerming teen oksidatiewe stres. Die resultate van ’n “microarray“ gebaseerde studie dui op ’n rol vir die sitokroom c heem liase (Cyc3p) in beide die beskermende en nadelige gevolge van karnitien aanvulling. Die vereiste vir sitochroom c dui op ’n moontlike rol vir apoptotiese prosesse. Hierdie hipotese word verder versterk deur ‘n analise van die impak van karnitien op genoomwye transkripsievlakke. ’n Afsonderlike doelwit van hierdie studie was toegespits op die klonering en uitdrukking van die vier ensieme betrokke in eukariotiese karnitien biosintese in S. cerevisiae. Die gekloneerde gene, uitgedruk vanaf die konstitutiewe PGK1 promotor, was geïntigreer in die gisgenoom om die pad op te bou. Die resultate van ’n plaat gebaseerde karnitien produksie toets dui aan dat die geneties gemanipuleerde gisrasse wel in staat is om trimetiellisien oor te skakel in Lkarnitien. Hierdie werk vorm die hoeksteen van ’n studie wat die ontwikkeling van karnitien produserende kommersiële gisrasse as doelwit stel.

Traumatic injuries to the spinal cord and brachial plexus induce a significant inflammatory response in the nervous tissue with progressive degeneration of neurons and glial cells, and cause considerable physical and mental suffering in affected patients. This thesis investigates the effects of the antioxidants N-acetyl-cysteine (NAC) and acetyl-L- carnitine (ALC) on the survival of motoneurons in the brainstem and spinal cord, the expression of pro-apoptotic and pro-inflammatory cell markers, axonal sprouting and glial cell reactions after spinal hemisection in adult rats. In addition, a novel MRI protocol has been developed to analyse the extent of neuronal degeneration in the spinal cord. Rubrospinal neurons and tibial motoneurons were pre-labelled with the fluorescent tracer Fast Blue one week before cervical C3 or lumbar L5 spinal cord hemisection. The intrathecal treatment with the antioxidants NAC (2.4mg/day) or ALC (0.9 mg/day) was initiated immediately after injury using Alzet2002 osmotic mini pumps. Spinal cord injury increased the expression of apoptotic cell markers BAX and caspase 3, induced significant degeneration of rubrospinal neurons and spinal motoneurons with associated decrease in immunoreactivity for microtubule-associated protein-2 (MAP2) in dendritic branches, synaptophysin in presynaptic boutons and neurofilaments in nerve fibers. Immunostaining for the astroglial marker glial fibrillary acidic protein and microglial markers OX42 and ED1 was markedly increased. Treatment with NAC and ALC attenuated levels of BAX, caspase 3, OX42 and ED1 expression after 2 weeks postoperatively. After 4-8 weeks of continuous intratheca ltreatment, NAC and ALC rescued approximately half of the rubrospinal neurons and spinal motoneurons destined to die, promoted axonal sprouting, restored the density of MAP2 and synaptophysin immunoreactivity and reduced the microglial reaction. However, antioxidant therapy did not affect the reactive astrocytes in the trauma zone. The inflammation modulating properties of ALC were also studied using cultures of human microglial cells. ALC increased the microglial production of interleukin IL-6 and BDNF, thereby possibly mediating the anti-inflammatory and pro-regenerative effects shown in vivo. To study degeneration in the spinal cord following pre-ganglionic and post-ganglionic brachial plexus injuries, adult rat models of ventral root avulsion and peripheral nerve injury were used. A novel MRI protocol was employed and the images were compared to morphological changes found in histological preparations. Ventral root avulsion caused degeneration of dendritic branches and axonal terminals in the spinal cord, followed by significant shrinkage of the ventral horn. Extensive astroglial and microglial reactions were detected in the histological preparations. Peripheral nerve injury reduced the density of dendritic branches but did not cause shrinkage of the ventral horn. Quantitative analysis of MRI images demonstrated changes in the ventral horn following ventral root avulsion only, thus validating the developed MRI technique as a possible tool for the differentiation of pre-ganglionic and post-ganglionic nerve injuries.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Master of Science / Department of Animal Sciences and Industry / Joel DeRouchey / Mike Tokach / Six experiments using 3,862 pigs were conducted to evaluate effects of ractopamine HCl (RAC) feeding programs, dietary L-Carnitine and dried distillers grains with solubles (DDGS) on growth, carcass traits, loin and jowl fat quality of pigs, and energy and protein sources in nursery diets. In Exp. 1 and 2, RAC-fed pigs had greater (P<0.05) ADG, G:F and HCW compared with the control. Within RAC treatments, there were no differences in growth. Pigs fed step-up RAC had increased (P<0.01) percentage lean, fat-free lean index and loin depth but decreased (P<0.01) backfat than the control or constant treatment. In Exp. 2, pigs fed step-up RAC program had greater (P<0.05) ADG and G:F than the constant treatment. Pigs fed constant RAC had greater (P=0.002) carcass yield than controls. There were no overall differences in other carcass traits among treatments. In Exp. 3, dietary L-Carnitine improved (P<0.02) ADG and final BW. A DDGS × L-Carnitine interaction (quadratic, P<0.01) was observed for G:F. Pigs not fed DDGS had similar G:F, but in DDGS diets pigs fed 50 ppm L-Carnitine had worse G:F than those fed 100 ppm. Pigs fed L-Carnitine had greater (P<0.02) HCW compared with those not fed L-Carnitine. Increasing L-Carnitine up to 100 ppm increased HCW (quadratic, P<0.03) and backfat (quadratic, P<0.04), with the maximum response at 50 ppm dietary L-Carnitine. Increasing L-Carnitine increased (linear, P<0.04) purge loss of loin. Feeding DDGS increased (P<0.001) linoleic acid and iodine value of jowl fat compared with feeding no DDGS. However, feeding L-Carnitine did not change jowl fatty acid composition. In Exp. 4, 5 and 6, nursery pigs fed choice white grease (CWG) had improved (P<0.02) G:F than pigs fed a control diet or an alcohol based energy source. Also, pigs fed CWG had greater (P<0.04) ADG in Exp. 4 and 6 and had reduced (P<0.01) ADFI in Exp. 5. The alcohol based energy source improved (P<0.04) ADG and ADFI with no change in G:F in Exp. 4; but did not affect growth in Exp. 5 and 6. In Exp. 6, pigs fed AV-E Digest had equal performance as nursery pigs fed other specialty proteins.

Introdução: A acidemia 3-hidroxi-3-metilglutárica é causada pela deficiência da 3-hidroxi-3-metil-glutaril-CoA-liase, uma enzima do metabolismo da leucina, levando ao acúmulo, especialmente, do ácido 3-hidroxi-3-metilglutárico nos tecidos. Estudos sugerem que o estresse oxidativo pode contribuir para os danos neurológicos observados em algumas acidúrias orgânicas. Objetivo: Avaliar parâmetros de estresse oxidativo em pacientes com acidúria 3-hidroxi-3-metilglutárica antes e após o tratamento. Materiais e Métodos: Amostras de sangue e urina foram coletadas de pacientes no momento do diagnóstico e após tratamento com dieta com restrição de proteínas e suplementação de L-carnitina (100mg/kg/dia) e de controles. O TBA, um subproduto final da peroxidação lipídica, foi medido no plasma. A determinação do teor de carbonilas e de grupos sulfidrila, marcadores de dano oxidativo a proteínas, foi realizada no plasma. Para avaliar na urina a oxidação de proteínas, os níveis de di-tirosina foram medidos por autofluorescência. O ensaio da capacidade antioxidante urinária foi realizado utilizando um kit comercial. Os níveis de carnitina livre e isovalerilcarnitina foram analisados em amostras de sangue por espectrometria de massas em tandem usando o método de monitorização de reação múltipla (MRM). A concentração de proteínas foi determinada pelo método de biureto em amostras de plasma usando um kit comercial. Resultados e Discussão: Os resultados demonstraram um aumento significativo nos níveis de isovalerilcarnitina em sangue total, das concentrações plasmáticas de malondialdeído e urinárias de di-tirosina, além de uma redução significativa da capacidade antioxidante urinária e dos níveis sanguíneos de carnitina livre nos pacientes no momento do diagnóstico em relação aos controles. Verificou-se uma diminuição nas concentrações do malondialdeído plasmático e da di-tirosina na urina dos pacientes tratados, o que sugere um efeito de proteção do tratamento sobre a peroxidação de lípidos e do dano oxidativo a proteínas, bem como uma normalização dos níveis de L-carnitina durante o tratamento. Conclusões: Esses resultados permitem sugerir que o estresse oxidativo ocorre em pacientes com acidemia 3-hidroxi-3-metilglutárica e que o tratamento com a dieta restrita de proteína e suplementada com L-carnitina pode oferecer proteção contra o dano oxidativo a biomoléculas. / Introduction: The 3-hydroxy-3-methylglutaric acidemia is caused by the deficiency of 3-hydroxy-3-methyl-glutaryl-CoA lyase, an enzyme of leucine metabolism, leading to accumulation of 3-hydroxy-3-methylglutaric acid in tissues. Studies have suggested that oxidative stress may contribute to the neurological damage observed in some organic acidurias. Objective: Evaluate oxidative stress parameters in patients with 3-hydroxy-3-methylglutaric aciduria patiets before and after treatment. Materials and Methods: Blood and urine samples were collected from patients at diagnosis and after treatment with restricted protein diet and supplemented with L-carnitine (100mg/kg/dia) and from controls. TBA , an end subproduct of lipid peroxidation, was measured in plasma. Determination of carbonyl and sulphydryl content, biomarkers of oxidative damage to proteins, was done in plasma. To assess urine protein oxidation, levels of di-tyrosine were measured by autofluorescence. The assay of antioxidant urinary capacity was performed using a commercial kit. The levels of free carnitine and isovalerylcarnitine were analyzed in blood samples by tandem mass spectrometry using the method of multiple reaction monitoring (MRM). Protein content was determined by the biuret method for plasma samples using a commercial kit. Results and Discussion: The results demonstrated a significant increase of total blood isovalerylcarnitine, malondialdehyde plasma concentrations and di-tyrosine urinary levels and a significant reduction of the urinary antioxidant capacity and free-carnitine blood levels in pacients at diagnosis compared to controls. It was verified a decrease in plasma malondialdehyde concentrations and urinary di-tyrosine levels in treated patients, suggesting a protective effect of the treatment on lipid peroxidation and protein oxidative damage, as well as a normalization of L-carnitine levels during treatment. Conclusions: These results allow to suggest that oxidative stress occurs in 3-hydroxy-3-methyl-glutaryl-CoA lyase deficient patients and treatment with restricted protein diet and L-carnitine may offer protection against oxidative damage.

Obesity is the most common clinical disorder and is associated with various medical conditions in dogs. Appropriate dietary management potentially provides weight loss in a safe, healthy, and efficacious manner. In order to elucidate whether dietary fiber, carnitine, diacylglycerol (DAG), and low glycemic index (LGI) act on such dietary components, a series of studies was conducted: 1) the combination of dietary fiber/carnitine effect on short term (3 and 7 h) satiety and long term (6 weeks) canine weight loss, 2) the combination of dietary LGI/high glycemic index (HGI) starches and DAG/triacylglycerol (TAG) effect during a 9 week canine weight loss period, and 3) the DAG effect on triglyceride rich lipoprotein (TRL) metabolism isolated from canine plasma 3-4 h postprandially. The combination of dietary fiber/carnitine supplementation decreased both food and energy intake at 3 h post-feeding, suggesting that this combination diet provided 3 h post-meal satiety. This combination supplement also increased postprandial plasma B- hydroxybutyrate (BHB) at d 42 and body fat and weight loss at d 42 from baseline. This combination supplement did not alter plasma vitamin A distributions or concentrations although it contained high vitamin A as B-carotene. In the second study, the LGI diets resulted in a more pronounced body weight loss than the HGI diets due to lower diet digestibilities. These data are consistent with LGI diets decreasing metabolizable energy and consequently consuming less energy compared to the HGI diets. The DAG diets lowered postprandial plasma TAG at weeks 1 and 8 in and increased plasma BHB at week 8, suggesting an increase in fat oxidation. The combination of DAG/LGI decreased postprandial total cholesterol at week 8. Lipoprotein concentrations were not altered by diet types. Fasting lipoprotein lipase (LPL) and hepatic lipase (HL) activities were not affected by diets. In the final study, DAG ingestion decreased TRL and plasma TAG concentrations vs. TAG ingestion. The DAG enriched meal increased non-esterified fatty acid, monoacylglycerol, and 1,3-DAG and decreased TAG in TRLs which may be attributed to larger TRL particle size compared to the TAG meal. Consequently, the DAG derived TRLs showed increased affinity of core TAG for LPL and HL in vitro. Moreover, the intravenous injection of the DAG derived canine TRLs into mice underwent more rapid blood clearance associated with the greater hepatic uptake compared to the TAG derived TRL injection. In conclusion, the combination of dietary fiber/carnitine and DAG/LGI preferably reduced body weight and stimulated fat oxidation, which promotes overall weight loss. The postprandial plasma TAG lowering effect of DAG is the result, at least partially, from the efficient clearance of TRLs from blood circulation and their ability to act as a more efficient substrate for plasma lipolytic enzymes.