Clin Chem Lab Med 2006;44(8):983–986  2006 by Walter de Gruyter • Berlin • New York. DOI 10.1515/CCLM.2006.176 2006/91 Article in press - uncorrected proof Measurement of carnitine in hemodialysis patients – adaptation of an enzymatic photometric method for an automatic analyzer Roman Cibulka 1, *, Romana Siroka 1 , Ladislav Trefil 1 , Jaroslav Racek 1 and Eugenie Vesela 2 1 Institute of Clinical Biochemistry and Hematology, Charles University Medical Faculty and Faculty Hospital, Pilsen, Czech Republic 2 Dialysis Center EuroCare, Pilsen, Czech Republic Abstract Background: The main goal of this work was to describe the analytical characteristics of an enzymatic photometric test for carnitine determination and its automation using an Olympus analyzer. Methods: We used a test from Roche intended for manual processing and tried to apply it for use on an Olympus AU 400 analyzer. The analytical parameters of our modified technique were determined using external quality controls and kit controls, and by measurements in venous blood samples from 85 chronically hemodialyzed patients (before and after hemodialysis) and from 68 healthy blood donors serv- ing as controls. Results: A reference value for free carnitine was esti- mated parametrically as 40.1"17.8 mmol/L. The mean bias for eight control measurements was 5.1%. Sen- sitivity was calculated as the limit of quantification at 2.6 mmol/L. The intra-assay coefficient of variation was 2.4%. The inter-assay coefficient of variation was 8.3%. Analytical recovery was 101.8%, 99.5% and 95.4%. Conclusions: The main advantages of our automated method in comparison to the original manual method are the smaller amounts of samples, reagents and di- luents required and the shorter analysis time. As hemodialysis patients often suffer from carnitine defi- ciency, we conclude that its determination may be helpful for diagnostic verification. Clin Chem Lab Med 2006;44:983–6. Keywords: carnitine; enzymatic photometric test; he- modialysis. Introduction L-Carnitine (3-hydroxy-4-N-trimethylbutyric acid) is a molecule with several roles in metabolism. First, car- nitine allows the transport of long-chain fatty acids *Corresponding author: Roman Cibulka, Institute of Clinical Biochemistry and Hematology, Charles University Hospital, Alej Svobody 80, 304 60 Plzen ˇ , Czech Republic Phone: q420-377104266, Fax: q420-377104234, E-mail: cibulkar@fnplzen.cz from the cytosol into the mitochondrial matrix for b-oxidation. Secondly, it mediates the transfer of products of peroxisomal b-oxidation into mitochon- dria for final oxidation in the Krebs cycle. Other func- tions include modulation of the acyl-CoA/CoA ratio, storage of energy as acetylcarnitine, stimulation of carbohydrate oxidation and excretion of acyl groups as acylcarnitine (1–4). Carnitine intake is mainly through meat products, but the body can partially ful- fil its needs by biosynthesis in the liver, kidneys and brain (2, 3). Hemodialysis (HD) patients often have low carnitine concentrations in plasma and tissues due to impaired synthesis in the kidneys and a loss across the dialysis membrane during HD (5–7). It has been reported that a single HD session reduces plasma carnitine to approximately one-third of its predialysis level. While levels of total carnitine in plasma may be normal or even elevated, the concentration of free carnitine (CAR) is markedly reduced in HD patients (8). The lack of CAR in HD patients leads to dyslipidemia, lethargy, muscular weakness, hypotension, cardiac dysfunction and arrhythmias, and recurrent cramps (4, 9). It can be difficult to distinguish these symptoms of CAR deficiency from similar ones related to uremia and dialysis treatment (4). In this respect, laboratory deter- mination of CAR may be helpful. The main goal of this work was to describe the ana- lytical characteristics of an enzymatic photometric method for CAR determination and its automation using an Olympus analyzer. Materials and methods We used an enzymatic photometric test from Roche Diag- nostics (Mannheim, Germany). The principle of detection described in the package insert for the test is as follows (10, 11). CAR is acetylated to acetylcarnitine by acetyl-CoA in the presence of the enzyme carnitine acetyl transferase. The resulting CoA is acetylated back to acetyl-CoA in the pres- ence of adenosine-59-triphosphate (ATP) and acetate, cata- lyzed by the enzyme acetyl-CoA synthetase. This results in the formation of adenosine-59-monophosphate (AMP) and inorganic pyrophosphate. In the presence of ATP, supported by myokinase, AMP forms at twice the amount of adenosine- 59-diphosphate (ADP). This is converted in subsequent reac- tion into phosphoenol pyruvate in the presence of pyruvate kinase. The pyruvate formed is reduced to L-lactate by reduced nicotinamide adenine dinucleotide (NADH) in the presence of lactate dehydrogenase. The amount of NADH consumed during the reaction is equivalent to half the amount of CAR. NADH is the parameter quantified, based on its absorbance at 340 nm. Brought to you by | Karolinska Institute Authenticated Download Date | 5/24/15 10:32 AM