edoc

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

Vernez, Laurence. Analysis of carnitine and acylcaritines in biological fluids and application to a clinical study. 2005, Doctoral Thesis, University of Basel, Faculty of Science.

[img]
Preview
PDF
858Kb

Official URL: http://edoc.unibas.ch/diss/DissB_7322

Downloads: Statistics Overview

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.
Advisors:Krähenbühl, Stephan
Committee Members:Hopfgartner, Gérard and Thormann, Wolfgang
Faculties and Departments:05 Faculty of Science > Departement Pharmazeutische Wissenschaften > Pharmazie > Pharmakologie (Krähenbühl)
UniBasel Contributors:Krähenbühl, Stephan
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:7322
Thesis status:Complete
Number of Pages:152
Language:English
Identification Number:
Last Modified:22 Apr 2018 04:30
Deposited On:13 Feb 2009 15:19

Repository Staff Only: item control page