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

Abstract

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