E. Arrigoni Martelli

Metabolism and disposition of intravenously administered acetyl-L-carnitine in healthy volunteers

SummaryThe pharmacokinetics of acetyl-L-carnitine hydrochloride were investigated in 6 healthy volunteers of both sexes after i.v. injection of 500 mg of the drug, expressed as inner salt.Plasma concentrations and urinary excretion of acetyl-L-carnitine (A), L-carnitine (B) and total acid soluble L-carnitine fraction were evaluated over a period lasting from 24 h before to 48 h after the administration. Plasma concentrations of A increased quickly after administration and then declined reaching base values within 12 h.Conversely, plasma concentrations of B rose more slowly, reaching a peak in 30–60 min, and then declined to base values within 24 h. Most of the injected dose of acetyl-L-carnitine was recovered in the urine during the first 24 h after administration as B and A.Mean renal clearance of both A and B during the first 12 h after injection was higher than the base values, suggesting the presence of a saturable tubular reabsorption process which may counterbalance major changes occurring in plasma concentrations of L-carnitine pattern.

Effect of propionyl-L-carnitine in a rat model of peripheral arteriopathy: a functional, histologic, and NMR spectroscopic study.

SummaryPropionyl-L-carnitine (PLC) has been shown to exert beneficial effects in experimental models of peripheral arterial diseases, such as ergotamine-induced tail gangrene and bilateral femoral arteries occlusion in rats. These models, however, present some drawbacks. The present study was performed to determine whether repeated oral administration of PLC improves the functional, histologic, and metabolic parameters in rats with long-lasting chemically induced peripheral arteriopathy. Peripheral arteriopathy was induced by injecting Na laurate in both the femoral arteries of rats. The walking capacity of the animals (treadmill test) was evaluated at different times and up to 5 weeks after Na laurate injection. Histological examination of vessels and muscles was performed at the end of the experimental period (5 weeks). In separate experiments the level of high-energy phosphates was determined with31P NMR methodology in the leg muscles. Injection of Na laurate impaired (p<0.05) the walking capacity of rats, caused thickening of the intima and marked narrowing of the vasal lumen, and reduced the ATP and PCr levels in muscles by 42% and 25%, respectively. PLC given orally for 7 days at 30, 60, 120, and 250 mg/kg dose-dependently decreased the severity of walking capacity impairment by 19%, 41%, 64%, and 71%, respectively. Long-term administration (4 weeks) of PLC (60 and 250 mg/kg os) caused a significant improvement of walking capacity throughout the entire period. The improvement persisted 1 week after discontinuation of the treatment. The severity of the vascular and muscular damages was markedly reduced, particularly in animals treated with the highest dose. Alterations in ATP and PCr levels were significantly (p<0.05) diminished by PLC (120 mg/kg os) administered daily for 15 days starting 24 hours after Na laurate injection, or for 11 days starting 4 days after Na laurate. The dextro-isomer of the compound was completely inactive, and L-carnitine improved motor performance to a much lesser degree than an identical dose of PLC. It is suggested that the activity of PLC is linked to its metabolic effects on fatty acid oxidation, with consequent preservation of high-energy phosphate levels.

GABA‐activated Chloride Currents of Postnatal Mouse Retinal Ganglion Cells are Blocked by Acetylcholine and Acetylcarnitine: How Specific are Ion Channels in Immature Neurons?

The goal of this study was to clarify pharmacological properties of GABAA receptors in cells of the mouse retinal ganglion cell layer in situ. Spontaneous synaptic currents and responses to exogenous GABA were recorded from individual neurons in retinal whole mounts (postnatal days 1–3) or retinal stripe preparations (postnatal days 4–6). Drugs were applied by a fast local superfusion system. Current responses were measured with the patch‐clamp technique in the whole‐cell configuration. All cells responded to exogenous GABA (average EC50 and Hill coefficient: 16.7 μM and 0.95 respectively) and generated GABAergic synaptic currents in response to elevated KCI. GABA‐induced currents of retinal ganglion cells were blocked by bicuculline, picrotoxin and Zn2+, as well as strychnine, and increased by pentobarbital, clonazepam and 3α‐hydroxy‐5α‐pregnan‐20‐one. In some retinal ganglion cells GABA caused an increase in the frequency of spontaneous synaptic currents, which points to a partially depolarizing action of this traditionally inhibitory neurotransmitter in the neural retina. Our major observation is that acetylcholine and acetylcarnitine blocked or reduced GABAergic inhibitory postsynaptic currents and responses to exogenous GABA. This effect was seen in only a fraction of retinal ganglion cells and occurred in both the undesensitized and the desensitized state of the GABAA receptor. The block was voltage‐independent and persisted during coapplication with the nicotinic and muscarinic acetylcholine receptor antagonists D‐tubocurarine and atropine. In contrast to GABA‐activated Cl− currents, glycine‐activated Ch currents remained unaffected by acetylcholine and acetylcarnitine. Acetylcarnitine had no effect on voltage‐activated Ca2+ channel currents and glutamate‐activated currents. Similar results were obtained in a dissociated cell culture preparation from the neonatal rat superior colliculus. In these cells acetylcholine induced a rightward shift in the dose ‐ response curve for GABA. Taken together, these results indicate that acetylcholine and acetylcarnitine can act directly at the GABAA binding site and thereby reduce the action of GABA in the immature retina.

Determination of aliphatic amines by gas and high-performance liquid chromatography

Abstract Aliphatic amines, such as mono-, di- and trimethylamine (MMA, DMA, TMA) and trimethylamine N-oxide (TMAO), are produced by bacterial degradation of trimethylalkylammonium compounds, such as choline, acetylcholine, carnitine and γ-betaines. This degradation can be environmental, intestinal or faecal1.2. This paper describes an analytical investigation by gas chromatography (GC) with flame ionization detection (FID) or thermionic specific detection (TSD) and by high-performance liquid chromatography (HPLC) with conductimetric, refractive index (RI) and electrochemical detection (ED) for the simultaneous determination of the above amines, mainly applied to biological samples such as urine, and an assay procedure adopted consisting in chemical reduction of TMAO to TMA, followed by evaluation of the latter by GC in order to determine the TMAO.

Elevated urinary excretion of endothelins in streptozotocin diabetic rats

Endothelin-1,2 urinary excretion, has been determined in control and streptozotocin diabetic rats at different times after diabetes induction. Diabetic rats showed increased urinary excretion of endothelins as compared to control rats, already three days after diabetes induction and up to 20 weeks.

Chromatographic and non-chromatographic assay of L-carnitine family components.

L-Carnitine and its acyl esters constitute an endogenous pool of the L-carnitine family, involved in the uptake of free fatty acids in the mitochondria by transfer across their membrane of the acyl moieties to fuel the beta-oxidation and the release of the acetyl group from the mitochondria to the cytosol. Therefore acyl-L-carnitine and acyl-L-carnitine transferase are involved in a homeostatic equilibrium with the cells. As most of these substances need to be monitored in foods, chemical and pharmaceutical processes and biological fluids, an overview of the main methods for assaying them is provided here, with specific reference to the intrinsic performance of each analytical procedure and with suggestions on the correct storage and manipulation of analytical samples.

L-CARNITINE AND CARNITINE ESTER TRANSPORT IN THE RAT SMALL INTESTINE

L-carnitine and its esters (acetyl-L-carnitine and propionyl-L-carnitine) at pharmacological doses (1, 5 and 10 mM) are absorbed by the rat jejunum by simple diffusion. Partition coefficients of carnitine esters determined in lipophilic media (diethyl ether/water and olive oil/water) are greater than that of L-carnitine. It would therefore seem that esters diffuse more easily through the lipid component of the intestinal barrier. The transport of acetyl- and propionyl-L-carnitine at pharmacological doses seems to be linearly and positively correlated with K+ transport but not with Na+ transport.

ENDOTHELINS URINARY EXCRETION IS INCREASED IN SPONTANEOUSLY DIABETIC RATS : BB/BB

Previously we reported (1) an increase of endothelin-1,2 (ET) content, in urine of rats made diabetic with streptozotocin (STZ), starting three days and up to 20 weeks from diabetes induction. The increased ET excretion was considered as an early marker of endothelial damage. To ascertain if this phenomenon was present also in a strain of spontaneously diabetic rats, endothelin-1,2 urinary excretion was determined in BB/BB diabetic rats, and their control (BB/WB), at different times after the onset of diabetes, (two, four, six and twelve weeks). BB/BB diabetic rats showed elevated urinary excretion of endothelins as compared to BB/WB control rats, starting two weeks after diabetes onset, and up to twelve weeks. In the same animals, Nerve Conduction Velocity (NCV), was monitored at the same time as an index of the occurrence of a diabetes complication (peripheral neuropathy). NCV resulted to be impaired in the BB/BB diabetic rats as compared to control rats; however the increase of ET in urine, is earlier in comparison to peripheral neuropathy. These data suggest the hypothesis that endothelial damages preceed the overt manifestations of peripheral neuropathy associated to diabetes.

Application of high-performance liquid chromatography to the analysis of propionyl-L-carnitine by a stereospecific enzyme assay.

Propionyl-L-carnitine was converted by chemical hydrolysis (0.3 M potassium hydroxide, pH 12.8, room temperature) into L-carnitine, which competes with crotonoylbetaine formation and can be quantitatively evaluated by an enzyme assay. Under the conditions selected, hydrolysis of propionyl-L-carnitine to L-carnitine was completed in a few minutes with less than 0.5% of crotonoylbetaine being formed. The method described is enantioselective and possesses the analytical requirements for assaying propionyl-L-carnitine during chemical synthesis procedures and in pharmaceutical formulations.

Some pharmacokinetic considerations about homeostatic equilibrium of endogenous substances

SummaryEndogenous substances in the body are controlled through simple, very effective mechanisms, that preserve an optimum homeostatic equilibrium of baseline concentration and restore it when impaired. When planning a pharmacokinetic investigation of an endogenous substance exogenously administered, it is imperative to carefully ascertain the above mechanisms as well as the baseline value and their possible variations associated with daily rhythm, food, age, sex, menstrual cycle.Often the control mechanisms operate through non-linear processes, therefore a non-compartmental analysis or a tailored model may be more appropriate than the compartmental models used in standard pharmacokinetic analysis.Some specific examples of endogenous substances are discussed here on the basis of the data from the literature and personal experience.