Heinz Löster

Stimulation of the anaerobic growth ofSalmonella typhimurium by reduction ofl-carnitine, carnitine derivatives and structure-related trimethylammonium compounds

In view of the development of al-carnitine deficiency, the metabolism ofl-carnitine and structure-related trimethylammonium compounds was studied inSalmonella typhimurium LT2 by means of thin-layer chromatography (TLC).l-Carnitine, crotonobetaine and acetyl-l-carnitine stimulated the anaerobic growth in a complex medium significantly. The stimulation depended on the formation of γ-butyrobetaine. The reduction ofl-carnitine proceeded in two steps: (1) Dehydration of thel-carnitine to crotonobetaine, (2) hydrogenation of crotonobetaine to γ-butyrobetaine. The reduction of crotonobetaine was responsible for the growth stimulation. Terminal electron acceptors of the anaerobic respiration such as nitrate and trimethylamine N-oxide, but not fumarate, suppressed the catabolism ofl-carnitine completely. Glucose fermentation, too, inhibited the reduction ofl-carnitine but optimal growth with a high carnitine catabolism was achieved byd-ribose. The esters of carnitine with medium- and long-chain fatty acids inhibited the growth considerably because of their detergent properties.

Determination of urinary acylcarnitines by ESI‐MS coupled with solid‐phase microextraction (SPME)

The determination of selected short-, medium- and long-chain acylcarnitines by electrospray ionization mass spectrometry (ESI-MS) is discussed. The differences in fragmentation behaviour and ionization efficiency are described in dependence on collision induced dissociation (CID) conditions and mixture composition. A new method combination, solid-phase microextraction (SPME)-ESI-MS, is introduced to characterize acylcarnitines in body fluids. This method utilizes SPME for pre-concentration of acylcarnitines from complex biological samples and ESI-MS for a selective and sensitive detection. The method is presented by standard experiments determining of acylcarnitines in aqueous solutions and urine samples from patients with secondary carnitine deficiency syndromes or related disorders.

L-carnitine reduces malondialdehyde concentrations in isolated rat hearts in dependence on perfusion conditions

The study investigated the influence of L-carnitine on the formation of malondialdehyde, an indicator of lipid peroxidation, in isolated Langendorff rat hearts. Earlier investigations of hemodynamic parameters and the recovery of ATP and creatine phosphate, carried out by means of 31P-NMR spectroscopy, had demonstrated that, depending on the composition of the perfusates (content of glucose, fatty acids, and carnitine), quite strong differences may occur in the reperfusion period after ischemia.In order to determine a possible relationship between these differences and the addition of carnitine, the study investigated whether carnitine penetrated into the tissue during the experiments, and whether it was able to reduce the concentration of detrimental substances. The concentrations of free and total carnitine as well as the malondialdehyde content as an indicator of ischemia/reperfusion damage were determined in different parts of the cardiac tissue as follows: After the Langendorff-experiments the hearts were dissected, homogenized and reconditioned; then carnitine and malondialdehyde were determined. The study included 63 hearts, which were divided into 8 different perfusion groups.Carnitine concentrations in heart tissue perfused with L-carnitine were much higher than those of the controls. Since exogenous L-carnitine and formed esters could be found in the tissue after the experiment, they must have permeated the cellular membrane rapidly. The concentrations of malondialdehyde behaved in an inverted way; as expected they were lower in carnitine-perfused hearts. The favourable effects of L-carnitine, expressed both by improved cardiac dynamics and ATP and CrP recovery in the reperfusion period, are obviously due to the fact that L-carnitine reduces ischemic damage.

Prolonged oral L-carnitine substitution increases bicycle ergometer performance in patients with severe, ischemically induced cardiac insufficiency

Summary. Acute and chronic L-carnitine application exerts protective effects in a number of cardiac diseases. These favourable effects are attributed to improvements of the energy metabolism and have been found both in animal experiments and in man. In order to investigate the effect of long-time oral L-carnitine substitution on physical performance, 41 patients suffering from class NYHA II or III cardiac insufficiency were recruited for a clinical study. Following the double-blind, randomized, placebo-controlled design of the study, 20 patients were given 3 × 1g L-carnitine daily for 120 days whereas the control group (21 patients) received placebo. Bicycle ergometer tests were used to determine maximum performance, systolic and diastolic blood pressure, heart rate, and ST changes. Four series of tests were carried out: on day 0 (before the first substrate application), on the 60th and the 120th day (during L-carnitine or placebo application), and on the 180th day (60 days after the end of substitution). A significant improvement in performance (significantly higher maximum performance during bicycle ergometry) could be found within the carnitine group on the 60th and 120th day of L-carnitine application; and haemodynamical parameters showed a tendency to improve, too. These effects, which were attributed to L-carnitine, could be detected even 60 days after the end of substitution. No corresponding changes were found in the placebo group.The findings presented in this paper support suggestions of other authors that L-carnitine in combination with the usual medication (digitalis, β-blockers, calcium antagonists, nitrates) improves performance and effort tolerance in patients with cardiac insufficiency. Moreover, the findings suggest a favourable long-term effect, which lasts beyond the actual L-carnitine application, on the performance of patients with advanced cardiac insufficiency.

EFFECTS OF L-CARNITINE AND ITS ACETYL AND PROPIONYL ESTERS ON ATP AND PCR LEVELS OF ISOLATED RAT HEARTS PERFUSED WITHOUT FATTY ACIDS AND INVESTIGATED BY MEANS OF 31P-NMR SPECTROSCOPY

Abstract31P-NMR in vivo spectroscopy is a non-invasive and non-hazardous technique which investigates chemical composition and metabolism of living objects, for example by determining phosphocreatine (PCr) and ATP concentrations. In the present study we investigated the influence of L-carnitine, acetyl-L-carnitine and propionyl-L-carnitine on the energetic state of the Langendorff rat heart subjected to an ischemic period of 20 min followed by a reperfusion period of 60 min. To avoid an overlapping of the effects of fatty acids and glucose, the hearts were perfused with a Tyrode solution containing no fatty acids. Ischemia causes a rapid decrease in the PCr signal, followed by a decrease in the ATP signal after a prolonged period of ischemia. At the same time, a drastic increase in the Pi signal was observed. A partial recovery of the ATP and PCr signals was observed in the reperfusion period. With L-carnitine a markedly improved recovery of the high energy phosphates (e.g. increased PCr/Pi ratios) was found. With acetyl-L-carnitine this effect was enhanced in the first postischemic phase. It was followed, however, by a more rapid decrease in the PCr/Pi ratio in the late reperfusion period. The effect of propionyl-L-carnitine was not significantly improved in the first minutes of the reperfusion period, but during the whole reperfusion phase a stabilization of the PCr/Pi ratio was observed. Intracellular pH can be calculated from determination of the Pi-chemical shift. This shows that L-carnitine and its derivatives have a protective effect against intracellular pH decrease during ischemia. L-carnitine improves the energetic state of the heart, which leads to increased ischemia tolerance. Hearts under L-carnitine were able to tolerate up to four ischemia-reperfusion periods in succession, whereas the controls were not able to do so. These NMR results confirm the hypothesis that L-carnitine and its esters have a protective effect in the reperfusion period of the ischemic rat heart. This could be of importance for the treatment of ischemic cardiac diseases.

Effects of L-carnitine on mechanical recovery of isolated rat hearts in relation to the perfusion with glucose and palmitate

The purpose of this study was to investigate the effects of L-carnitine on the hemodynamic parameters of Langendorff hearts. Isolated rat hearts were perfused with various solutions containing high or low concentrations of fatty acids, additional glucose or no glucose, and L-carnitine or no L-carnitine. The most interesting part of the experiments was the behaviour of the hearts in the reperfusion period after no-flow ischemia of 20 min. The results were: (1) With glucose and high fatty acid concentrations the hearts showed an improved recovery of the left ventricular functions in the reperfusion period compared with low fatty acid concentrations. Without glucose the left ventricular pressure is much lower in the reperfusion period. (2) Addition of L-carnitine improved the recovery of the ischemically damaged hearts. This improvement is especially impressive at low fatty acid concentrations. L-carnitine addition at high fatty acid concentrations but without glucose strongly improved reperfusion behaviour. (3) The coronary flow is increased by 2 experimental conditions: (i) perfusion at low levels of fatty acids, carnitine and with glucose and (ii) high levels of fatty acids and carnitine but without glucose. These findings suggest that supplementation of L-carnitine has a beneficial effect on the isolated heart under various conditions, and possibly on specific human heart diseases.

Synthesis of [methyl-14C]crotonobetaine from DL-[methyl-14C]carnitine

The causes of carnitine deficiency syndromes are not completely understood, but decomposition of L-carnitine in vivo is likely to be involved. Carnitine is metabolized to γ-butyrobetaine, and crotonobetaine is probably an intermediate in this pathway. To validate experimentally the precursor-product relationship between the three physiologically occuring γ-betaines - L-carnitine, crotonobetaine, y-butyrobetaine - labelling with stable or radioactive isotopes became necessary. Methyl-labelled carnitine isomers (L(-)-, D(+)- or DL-) or γ-butyrobetaine can be easily synthesized by methylation of 4-amino-3-hydroxybutyric acid isomers or 4-aminobutyric acid, respectively. Because of problems with the 4-aminocrotonic acid, we synthesized labelled crotonobetaine from labelled carnitine. Thus, DL-[methyl- 14 C]carnitine was dehydrated by reaction with concentrated sulfuric acid. After removal of the latter the products were separated and purified by ion exchange chromatography on DOWEX 50 WX8 (200 - 400 mesh) and gradient elution with hydrochloric acid. In addition to the labelled main product (methyl- 14 C)crotonobetaine (yield about 50%), [methyl- 14 C]glycine betaine and [methyl- 14 C]acetonyl-trimethylammonium (ATMA) were formed. The end products were identified by combined thin layer chromatography/autoradiography and quantified by liquid scintillation counting.