Analysis of carnitine and acylcaritines in biological fluids and application to a clinical study

摘要: Carnitine, an endogenous compound present in most mammalian tissues, is involved the transport of activated fatty acids between cellular organelles and thus plays important role in acid metabolism energy production. Carnitine binds acids, generating various acylcarnitines with different chain lengths. also implicated in the maintenance the pool free coenzyme A elimination potentially toxic acyl-CoA. In mammals, carnitine provided for two thirds by dietary intake one third biosynthesis from amino L-lysine L-methionine. Since is present body tissues at much higher concentrations than plasma, transport systems ensure it’s widespread distribution sites absorption synthesis throughout the body. many metabolic disorders, greatly disturbed, leading to a redistribution acylcarnitine pools. The determination individual acylcarnitines biological fluids a powerful means diagnose these disorders. It was the aim this thesis work develop analytical tools and acylcarnitines fluids. Finally, one developed assay utilized follow up of clinical study. In chapter 1, current knowledge about acylcarnitines, including carnitine function, homeostasis, are reviewed. Cases deficiencies are discussed, description available methods used carnitine and completes introduction part. Chapter 2 describes capillary electrophoresis method profile carnitine, shortand medium-chain after solid-phase extraction on silica column. The assay enabled separation five standard solutions, in urine spiked urines, characterized acetylcarnitine in standard solutions. quantified urine samples results were compared with obtained using radio-enzymatic assay. Chapter 3 presents high-performance liquid chromatography coupled tandem mass spectrometry detection (HPLC-MS/MS) eight different acylcarnitines, long-chain acylcarnitines. Samples submitted solid-phase extraction cation-exchange column prior injection system. detection is performed mass spectrometry, derivatization not necessary. The separation achieved volatile ion-pair reagent. validation the determination both stable isotope derivative as internal water calibration matrix. results obtained quantification compared those a radio-enzymatic method. Application patients suffering different organic acidurias diagnosis disorders. The extension HPLC-MS/MS plasma samples, minor modifications protocol, protein precipitation, reported 4. Butyrobetaine, the direct precursor contrast urine, and could be analyzed during same analysis. Quantification acetylcarnitine, propionylcarnitine, isovalerylcarnitine, hexanoylcarnitine, octanoylcarnitine butyrobetaine were validated solutions 4% bovine serum albumin solution Serum patient methylmalonic aciduria successfully identified characteristic disorder. The concrete use illustrated 5. clinical study conducted 7 end-stage renal disease undergoing longterm hemodialysis. As efficiently removed hemodialysis session, leading reduced levels relative increase aim the study investigate composition pools in these patients, baseline conditions they supplemented the end each session. Extraction kinetics session and kinetics intravenous administration studied. A comparison established when given either no supplement or two different dosages carnitine. supplementation corrected hypocarnitinemia and yielded increased suggesting that substitution in hemodialysis could useful removal potentially toxic acyl-groups.