A convenient method for laboratory synthesis of the N-([1-14C]palmitoyl)ethanolamine, labeled by a fatty acid

Data concerning the synthesis of bioactive lipid compound N-([1-14C]-palmitoyl)ethanolamine labeled by 14C fatty acid are reported. The method is based on the ability of ethanolamine and fatty acid to the direct chemical condensation at 180 degrees C with yielding of N-acylethanolamine. The purification of the end product by the double crystallization in ethanol allows to obtain chromatographically pure substance. The presented method of the labeled N-([1-14C]-palmitoyl)ethanolamine synthesis is simple and in extensive that's why it might be used in the area of biologically active compounds investigation.     

Stearoyl[1-14C]sulfogalactosylsphingosine ([14C]sulfatide) as substrate for cerebroside sulfatase assay

A convenient method for large scale preparation of stearoyl[1-¹⁴C]sulfogalactosylsphingosine has been developed. The first step consists in preparing the lysosulfatide intermediate with a good yield, which we have successfully performed. In the second step, the lysoderivative is coupled to [1-¹⁴C]stearic acid through the acyl chloride procedure. The labeled substrate thus synthetized appears to be particularly convenient for cerebroside sulfatase determination, because of the stability of the ¹⁴C isotope, compared to the other isotopes (tritium or ³⁵S) which have been previously used for the same purpose.     

A comparison of the brain uptake of N-(cyclopropyl[11C]methyl)norbuprenorphine ([11C]buprenorphine) and N-(cyclopropyl[11C]methyl)nordiprenorphme ([11C]diprenorphine) in baboon using PET

Buprenorphine and diprenorphine were radiolabeled with ¹¹C and their distributions in the baboon brain were studied using positron emission tomography (PET). Specific binding was demonstrated in the striatum (but not in the cerebellum) by pretreating the baboon with (−)naloxone. The absolute striatal uptakes and time courses were similar for these two radioligands but the ratio of radioactivity in the striatum to cerebellum in the baboon was higher for [¹¹C]diprenorphine than for [¹¹C]buprenorphine. Analysis of baboon plasma indicated that both [¹¹C]diprenorphine and [¹¹CJbuprenorphine are rapidly metabolized. Analysis of radioactivity in mouse brain indicated that these two radioligands are stable to metabolic transformation. At 30 min after injection, 86–90% of extracted radioactivity was due to unchanged ¹¹C-labeled radioligands. These results suggest that both [¹¹C]diprenorphine and [¹¹C]buprenorphine may be useful radioligands for studying opioid receptors in humans, although [¹¹C]diprenorphine may be a better radioligand than [¹¹C]buprenorphine for this purpose because of its more rapid clearance from the cerebellum.     

Comparison of [3H] phencyclidine ([3H] PCP) and [3H] N-[1-(2-thienyl) cyclohexyl] piperidine ([3H] TCP) binding properties to rat and human brain membranes

The investigation of [3H] PCP and [3H] TCP binding properties to rat cerebrum and cerebellum resulted in the demonstration of multiple binding sites for the two drugs. In the two tissue preparations PCP had a lower affinity than TCP. In membranes from the cerebrum an equal number of high affinity binding sites were present for [3H] PCP and [3H] TCP. However, low affinity binding sites were two times more numerous for [3H] PCP than for [3H] TCP. In the cerebellum, the number of high and low affinity sites labeled by the two radioligands was identical, but the number of high affinity sites was about 7 fold lower than in the cerebrum. Taken together these results may indicate that in the cerebrum [3H] PCP labels other sites than NMDA/PCP receptor(s), maybe sigma receptors and/or the dopamine uptake complex. In human cerebral cortex samples [3H] TCP also bound to two different sites. The number of high and low affinity sites were 12 and 3 times, respectively, less abundant than in the rat cerebrum. Low affinity sites were of higher affinity (5 times) than corresponding sites in the rat brain. In the human cerebellum [3H] TCP binding parameters were identical to those measured in the same region in the rat.     

Synthesis and in vitro evaluation of (S)-2-([11C]methoxy)-4-[3-methyl-1-(2-piperidine-1-yl-phenyl)-butyl-carbamoyl]-benzoic acid ([11C]methoxy-repaglinide): a potential beta-cell imaging agent

The 11C-labeled sulfonylurea receptor 1 (SUR1) ligand (S)-2-([11C]methoxy)-4-[3-methyl-1-(2-piperidine-1-yl-phenyl)-butyl-carbamoyl]-benzoic acid ([11C]methoxy-repaglinide) was synthesized in an overall radiochemical yield of 35% after 55 min with a radiochemical purity higher than 99%. This compound is considered for the noninvasive investigation of the SUR1 receptor status of pancreatic beta-cells by positron emission tomography (PET) in the context of type 1 and type 2 diabetes. The specific activity was 40-70 GBq/micromol. In vitro testing of the nonradioactive methoxy-repaglinide was performed to characterize the affinity for binding to the human SUR1 isoform. Methoxy-repaglinide induced a complete monophasic inhibition curve with a Hill coefficient close to 1 (1.03) yielding a dissociation constant (KD) of 83 nM and an IC50 of 163 nM. Insulin secretion experiments on isolated rat islets were performed to prove biological activity, which was determined to be in the same range as that of original repaglinide.     

The effect of metyrapone on cytokinin ([8-14C]benzylaminopurine) metabolism in mature green tomato pericarp

Cytokinin, [8-14C]Benzylaminopurine, metabolism in tomato pericarp was followed during a 3 h period utilizing thin layer chromatography and visualization by fluorography. Fluorography indicated the formation of at least 7 metabolites during 3 h. Cytokinin metabolism was reduced by approximately 40% in 3 h by the presence of 250 microM metyrapone, an inhibitor of cytochrome P-450 related enzyme systems. In the presence of metyrapone, the number of radioactive metabolites on the thin layer plate was reduced from 7 to 4 and 2 of these were unique to the metyrapone-treated sample. These data suggest the initial step in benzylaminopurine metabolism in tomato pericarp may be mediated by a cytochrome P-450 related enzyme system which is altered in the presence of metyrapone.     

Effect of insulin and cortisol on oxidation of (1-14C)glucose, (6-14C)glucose, (1-14C)palmitate and (1-14C)leucine in the tissues of swine during the neonatal period

The intensity of [1-14C]glucose, [6-14C]glucose, [1-14C]palmitate and [1-14C]leucine oxidation and the effect of insulin and hydrocortisone on this process were studied in the brain, duodenum mucosa, liver and skeletal muscle of 1- and 5-day old piglets in vitro. Most of the studied substrates are oxidized in the tissues of 5-day piglets more intensively. Insulin stimulates oxidation of [1-14C]glucose, [6-14C]glucose and [1-14C]leucine in the brain and duodenum mucosa in 1- and 5-day old piglets, while in the liver and skeletal muscle--only in 5-day old piglets. Hydrocortisone administration enhances oxidation of [1-14C]leucine in most of the studied tissues in 1-day piglets and oxidation of [1-14C]glucose and [6-14C]glucose--in 5-day piglets. Both hormones produce no essential influence on the intensity of [1-14C]palmitate oxidation in the studied tissues of piglets or somewhat weaken it.     

Metabolism of [1-14C]glyoxylate, [1-14C]-glycollate, [1-14C]glycine and [2-14C]-glycine by homogenates of kidney and liver tissue from hyperoxaluric and control subjects

1. The metabolism of [1-(14)C]glyoxylate to carbon dioxide, glycine, oxalate, serine, formate and glycollate was investigated in hyperoxaluric and control subjects' kidney and liver tissue in vitro. 2. Only glycine and carbon dioxide became significantly labelled with (14)C, and this was less in the hyperoxaluric patients' kidney tissue than in the control tissue. 3. Liver did not show this difference. 4. The metabolism of [1-(14)C]glycollate was also studied in the liver tissue; glyoxylate formation was demonstrated and the formation of (14)CO(2) from this substrate was likewise unimpaired in the hyperoxaluric patients' liver tissue in these experiments. 5. Glycine was not metabolized by human kidney, liver or blood cells under the conditions used. 6. These observations show that glyoxylate metabolism by the kidney is impaired in primary hyperoxaluria.     

Distribution of ]1-14C]N-palmitoylethanolamine and its metabolites in subcellular fractions of neuroblastoma C1300 N18

Distribution in dynamics of [1-14C]N-palmitoyl ethanolamine in the subcellular fractions of neuroblastoma C1300 N18 has been studied. It is shown that distribution dynamics of the label is different depending on subcellular fractions. The level of the label in cytoplasm 15 min after incubation reaches the value which remains constant till the end of the experiment. In the microsomal fraction the label is accumulated with time and becomes maximum at the end of the experiment. The highest amount of the label in the plasma membrane has been found 15 min later and then its amount falls. Parallel with this the amount of free fatty acids grows and then begins to fall simultaneously with an increase of the amount of esterified fatty acids. A considerable amount of the label of fatty acids has been determined 15 min later in the composition of di- and triglycerides of cytosol and microsomal fraction.     

Metabolism of (3-(14)C)tryptophan and (2-(14)C)alanine in cattle tissues

In the experiments involving incubation of the liver, brain cortex, muscle and adipose tissues homogenates with [3-14C] tryptophan for an hour 43.2-89.3% of the label was found in proteins, 7.2-47.2%--in lipids, 2.6-9.4%--in CO2. Following incubation of the above-mentioned tissue homogenates with [2-14C] alanine, proteins, lipids and CO2 contain 28.8-49.3%; 22.6-31.9% and 21.6-49.3% of radioactive label, respectively. Radioactivity of lipids synthesized by the homogenates of the investigated tissues from [3-14C] tryptophan and [2-14C] alanine is 23.5-63.5 and 21.1-56.0%, respectively, the radioactivity of CO2 being 1.4-5.1 and 9.3-11.8% of the above-mentioned compounds synthesized from [1-14C] acetate. The results obtained testify to the considerable contribution of [3-14C] tryptophan and [2-14C] alanine to protein synthesis as well as to their involvement in the substrate supply of lipogenesis and energetic processes in various organs and tissues of cattle.