Interference of heparin in plasma levocarnitine determination with radioenzyme assay.

This paper describes the interference of heparin (CAS 9005-49-6) in whole plasma used for free and total levocarnitine (L-carnitine, CAS 541-15-1) analysis. Alkaline hydrolysis required for total L-carnitine measurement does not overcome the problem. The same interference is also present in long-chain acyl-L-carnitine assay, because heparin precipitates together with proteins and long-chain esters of L-carnitine in the acid insoluble fraction of perchloric acid treated plasma sample. In this case an appropriate cleaning of the sample is recommended before the assay.

Plasma concentration, urinary excretion and renal clearance of L-carnitine during pregnancy: a reversible secondary L-carnitine deficiency.

Plasma concentration, urinary excretion and renal clearance of free, total and esterified L-carnitine were monitored monthly in 14 women during the last 6 months of pregnancy and 1 month after delivery. Plasma concentration and renal clearance measured 1 month after delivery overlapped with normal values for females of comparable age, and were considered the reference values for further comparisons. Plasma concentration of free, total and esterified L-carnitine decreased during pregnancy, reaching values as low as half of those measured 1 month after delivery, whereas urinary excretion and renal clearance, mainly of L-carnitine esters, increased, with renal clearance reaching a peak at the 16th week of pregnancy. Pregnancy thus leads to a reversible secondary deficiency of L-carnitine. The involvement of L-carnitine in the excretion of an excess of acyl-S-coenzyme A groups to prevent a possible systemic acidosis, as well as hormonal changes and a reduction of L-carnitine biosynthesis, could play a significant role in the variations in L-carnitine metabolism encountered in pregnancy. As physiological components of L-carnitine are excreted via a saturable tubular reabsorption, their threshold seems to follow plasma concentration, even when they decrease markedly, as in pregnancy.

Effect of acetyl-L-carnitine treatment on the levels of levocarnitine and its derivatives in streptozotocin-diabetic rats.

The effect of diabetes induced by streptozotocin and that of acetyl-L-carnitine (ALC) hydrochloride (CAS 5080-50-2) treatment on the homeostasis of the levocarnitine (L-carnitine) moiety was investigated in Sprague-Dawley rats. The diabetic status was ascertained by measuring blood glucose. L-carnitine (LC), total acid soluble L-carnitine (TC) and ALC were measured in serum, tissues and urine by radioenzymatic methods. Short-chain L-carnitine esters (SCLCE) were obtained by subtracting LC from TC. Serum concentration of L-carnitine moiety was decreased in diabetic when compared to normal rats; whereas ALC oral treatment (50 and 150 mg/kg p.o. for 4 weeks) in diabetic rats increased, dose-dependently, all the components of L-carnitine moiety, SCLCE and ALC being completely restored. In the liver of diabetic rats all the analytes proved to be higher than in normal rats, mainly LC and TC. A similar trend was observed in skeletal muscle, at least with LC and TC, whereas SCLCE and ALC were not affected. The treatment with ALC increased the liver concentration of all the analytes in a dose-related way whereas in skeletal muscle only LC and TC showed an increase with the highest dose of ALC. Myocardium and kidneys showed a decrease of all the analytes in diabetes; the treatment with ALC normalized the situation in kidneys, in a dose-related way, but not in the myocardium. Urinary excretion and renal clearance of L-carnitine moiety increased in diabetes; an additional dose-related increase was observed with the ALC treatment.

Hemodynamic and metabolic activities of propionyl-L-carnitine in rats with pressure-overload cardiac hypertrophy.

Evidence has been put forth that a number of human and experimental cardiomyopathies are associated with a lower myocardial carnitine content. This study was performed to test the hypothesis that the correction of carnitine derivative, propionyl-L-carnitine (PLC), may improve cardiac function. Repeated administration of PLC was compared to saline with respect to cardiac function in rats with pressure-overload cardiac hypertrophy and low myocardial carnitine levels. Cardiac hypertrophy was induced by abdominal aorta constriction in rats. Separate groups of rats were used for (a) determination of myocardial carnitine content, (b) evaluation of in vivo hemodynamics, and (c) evaluation of performance and metabolic state of Langendorff perfused hearts. Results showed the following: (i) The myocardial carnitine content was inversely correlated to cardiac hypertrophy (r = 0.68, p less than 0.05) and PLC treatment (50 mg/kg i.a. for 4 days) restored it to normal values (ii) The PLC effect on cardiac function was significantly and directly related to cardiac hypertrophy [correlations between heart weight and percent changes in cardiovascular parameters: cardiac output (CO), p less than 0.001; cardiac work (CW), p less than 0.01, stroke volume (SV) and stroke work (SW), p less than 0.02]. In animals with heart weight greater than 1,400 mg, the effect of PLC on CO, CW, SV, SW, and total peripheral resistance (TPR) was significantly different from that of saline (CO, CW, SV, and SW, p less than 0.005 each; TPR, p less than 0.05). The effect was observed 24 h after the first PLC administration and significantly diminished following a 4 day suspension of the treatment. (iii) Perfused hearts from PLC-treated rats displayed a significantly lower left ventricular end-diastolic pressure (p less than 0.01) and greater relaxation rate (p less than 0.05) than those from control rats. Moreover, in PLC-treated hearts, the content of creatine phosphate, ATP, and total adenine nucleotides (ATP+ADP+AMP; TAN) was significantly increased (CP, p less than 0.05; ATP and TAN, p less than 0.01 vs. control). These data show that PLC exerts a stimulatory activity on hearts with hypertrophy and low carnitine content, implying that carnitine deficiency may contribute to the depression of cardiac function in this model.

Enzymes in stereoselective pharmacokinetics of endogenous substances.

The use of enzymes to assay individual components of the L-carnitine family in pharmaceuticals, foodstuffs, and biological fluids with various forms of detection is reviewed. The most useful enzyme in the assay of compounds of the L-carnitine family is carnitine acetyl transferase (CAT), which catalyses the reversible interconversion of L-carnitine and its short-chain acyl esters. CAT can be used in one or more coupled reactions combined with U.V., or radiolabelled detection, or combined with HPLC, allowing, enantioselective, structurally specific, and, in the case of radiolabelled tracing, highly sensitive assays to be carried out. When compared with chromatographic separation of enantiomers or diastereoisomers, enantioselective enzyme mediated assays may be cheaper, more sensitive, and simpler, but they do not allow the nonpreferred isomer to be assayed. Consequently, they are appropriate for the specific assay of endogenous enantiomeric substrates of the enzyme concerned, in biological samples. The analysis of the other enantiomer in raw materials or in pharmaceuticals must be more properly approached by enantioselective chromatographic methods.

Pharmacokinetic investigation of ST 789 in rats and mice.

Pharmacokinetics of ST 789 were investigated in rats and mice after oral, intravenous, subcutaneous and intramuscular routes. A HPLC method validated for pharmacokinetic studies allowed the Authors to assay ST 789 concentration in plasma, urine and tissues. ST 789 interacted poorly with albumin and plasma proteins. Blood-to-plasma concentration ratio proved to range on average from 1.3 to 2.0 in both in vivo and in vitro studies. Plasma concentration-time behaviour after i.v. injection fitted according to the open three-compartment model; after subcutaneous and intramuscular routes two phases were observed and after oral route the absorption and one elimination phases were detected. Pharmacokinetics of ST 789 proved to vary linearly with the dose administered. Cumulative urinary excretion after parenteral administration ranged on average 60-80% and cumulative biliary excretion was 9.47% of the dose given. Oral administration allowed only 2.5% of the drug given to be excreted in urine, this leading to conclude that this drug is poorly absorbed through the intestine wall. After oral administration ST 789 produced relatively high concentration in lungs and lymphatic tissues, this leading to hypothesize a lymphatic component in its enteral absorption.

High-performance liquid chromatographic evaluation of Med 15 and its metabolites Med 5 and tolmetin in rat plasma.

A simple and reliable high-performance liquid chromatographic method is described for the quantitative analysis of the new non-steroidal anti-inflammatory agent Med 15 and its metabolites Med 5 and tolmetin in rat plasma. After selective extraction the three analytes and an internal standard (p-phenyl-phenol) were separated on a reversed-phase Ultrasphere 5 micron column using potassium dihydrogenphosphate (0.05 M)-acetonitrile (52:48) (pH 4.7) as the mobile phase. The analytes were detected at 313 nm; the sensitivity of the method proved to be 0.05 microgram/ml for all three compounds. The method has been applied to investigate Med 15 pharmacokinetics in rats.

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.

Homeostatic equilibrium of L-carnitine family before and after i.v. administration of propionyl-L-carnitine in humans, dogs and rats.

Propionyl-L-carnitine is a minor component of L-carnitine family which, when exogenously administered, proved to possess interesting cardiovascular activities. In this paper the pharmacokinetics of propionyl-L-carnitine was investigated in humans, dogs and rats after intravenous administration. In all the three species the base homeostatic equilibrium was carefully investigated in plasma and in urine during the 24 h period before the administration. Propionyl-L-carnitine, acetyl-L-carnitine, L-carnitine and total acid soluble L-sensitive were assayed in plasma and urine with very sensitive enantioselective radioenzyme assay. After dosing propionyl-L-carnitine rapidly increased, and then decreased reaching the base value within 6 h or more, depending on the species and the dose. Also L-carnitine in all the three species and acetyl-L-carnitine in rats and dogs increased, but the increase was more sustained when compared to propionyl-L-carnitine. Urinary excretion paralleled plasma concentration, reaching the highest value in the 24 h-period after dosing. Renal clearance also increased reflecting the behaviour of plasma concentration and urinary excretion. Results obtained all the conclusion that two homeostatic equilibrium of L-carnitine family components, namely the inter-exchange between L-carnitine and its esters catalyzed by carnitine acyl transferases, and a saturable tubular reabsorption process with differentiated threshold for each component.

Protein binding of L-carnitine family components.

L-carnitine and its short-, medium- and long-chain acyl esters constitute the L-carnitine family. These compounds in the body are equilibrated according to a homeostatic equilibrium preserved and, when impaired, restored by a dynamic inter-exchange between L-carnitine and its esters, catalysed by carnitine acyl transferases, and a tubular reabsorption process with differentiated thresholds for each component. The interaction of these compounds with albumin and plasma proteins of rats, dogs and humans was carefully investigated by means of ultrafiltration and gel filtration techniques. Results obtained demonstrate that L-carnitine and its short-chain esters, namely acetyl-L-carnitine and propionyl-L-carnitine, do not interact with either albumin or plasma proteins; octanoyl-L-carnitine interacts in a measurable even if poor extent (12-30%), whereas palmitoyl-L-carnitine, a molecule with a detergent activity, is completely bound to albumin and plasma proteins.

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.

High-performance liquid chromatographic evaluation of PCF 39, a new immunomodulator agent.

An high-performance liquid chromatographic analysis of PCF 39, N2-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-arginine, with ultraviolet detection, has been devised and validated. The main pharmacokinetic results encountered for rats treated intravenously with PCF 39 at a dose of 100 mg/kg are described.

A radiometric assay for ganglioside sialidase applied to the determination of the enzyme subcellular location in cultured human fibroblasts.

A radiometric method for the assay of ganglioside sialidase in cultured human fibroblasts was set up. As substrate, highly radioactive (1.28 Ci/mmol) ganglioside GDla isotopically tritium-labeled at carbon C-3 of the long chain base was employed; the liberated, and TLC separated [3H]GM1 was determined by computer-assisted radiochromatoscanning. Under experimental conditions that provided a low and quite acceptable (4-5%) coefficient of variation, the detection limit of the method was 0.1 nmol of liberated GM1, using as low as 10 micrograms of fibroblast homogenate as protein. The detection limit could be lowered to 0.02-0.03 nmol, adopting conditions that, however, carried a higher analytical error (coefficient of variation over 10%). The content of ganglioside sialidase in human fibroblasts cultured in 75-cm2 plastic flasks was 5.8 +/- 2.5 (SD) nmol liberated GM1 h-1 mg protein-1. Subfractionation studies performed on fibroblast homogenate showed that the ganglioside sialidase was mainly associated with the light membrane subfraction that was rich in plasma and intracellular membranes. This subfraction displayed almost no sialidase activity on the artificial substrate 4-methylumbelliferyl-D-N-acetylneuraminic acid. A small but measurable ganglioside sialidase activity was also present in the lysosome-enriched subfraction, which contained a very high sialidase activity on the above artificial substrate. All this supports the hypothesis that human fibroblasts contain sialidases with different subcellular location and substrate specificity. Particularly, the sialidase acting on gangliosides seems to have two sites of subcellular location, a major one at the level of plasma membranes and/or intracellular organelles functionally related with the plasma membranes and a minor one in the lysosomes.

Pyruvate-dehydrogenase complex in ataxic patients: enzyme deficiency in ataxic encephalopathy plus lactic acidosis and normal activity in Friedreich ataxia.

Pyruvate dehydrogenase complex (PDHC) activity was measured in cultured fibroblasts from 12 patients with Friedreich's ataxia (FA), and in 1 patient with lactic acidosis and ataxia. The activities obtained after extraction of PDHC by different methods were compared. Triton-X-100 extraction yielded enzyme activities 5 to 10 times greater than those obtained with the older methods. With this sensitive technique, PDHC activity was markedly deficient in fibroblasts from the patient with lactic acidosis and ataxia but it was normal in the fibroblasts from FA patients. Mg++ activation of the PDHC in FA fibroblasts was normal.

Heterogeneity of carnitine-palmitoyltransferase deficiency.

Episodes with muscle ache, rhabdomyolysis and myoglobinuria with or without associated renal insufficiency are characteristic of muscle carnitinepalmitoyltransferase (CPT) deficiency. However, patients differ from each other in many aspects, such as the kind of stimulus that triggers rhabdomyolysis, the ability to produce ketone bodies when fasting, whether the enzyme defect is localized in skeletal muscle or is general, and the nature of the enzyme defect, which may be in CPT I or CPT II or both. Studies of muscle, liver and fibroblasts from a patient with recurrent rhabdomyolysis spontaneously occurring or triggered by exercise or fever, revealed a CPT deficiency in the muscle and liver biopsy samples but normal CPT activity in cultured cells, differing from previously reported patients. The enzyme defect in muscle was evidenced by two different methods, but not when determined with a method that measures the formation of palmitoylcarnitine. The enzyme abnormality in the patient's liver was associated with a delayed ketone body production and with a dramatic increase in long-chain acylcarnitines in the serum when fasting. Moreover the patient was unable to build up ketones when fed long-chain triglycerides (LCT) but showed prompt ketogenic response when fed medium-chain triglycerides (MCT). The heterogeneity of clinical presentations and of the biochemical findings in patients with CPT deficiency are discussed.