Thin-layer Radiochromatography Research Articles

Pharmacokinetics of 11C-BCNU in experimental brain tumor.

Wistar rats implanted intracerebrally with AA ascites tumor were injected seven or eight days later with 11C-labelled BCNU. Radioactive compounds in samples of plasma, tumor, and contralateral brain were identified after injection at intervals by doing chloroform extraction and thin layer radiochromatography. At 60 min after injection radioactivity levels were 56% higher in the tumor than in contralateral brain. This increase was due mainly to 2-chlorethyl isocyanate, which binds to amino groups and/or nucleic acid. The results demonstrated both a higher concentration and a faster decomposition of BCNU in the tumor.

Analysis of androgen-5&-reductase by an enzyme kinetic method

Analysis of 5&-reductase(5&R) is commonly done by measurement of distinct testosterone(T) metabolites, following tissue incubation. Interlaboratory comparison is difficult, as different individual methods are used. In order to overcome this disadvantage, 5&R analysis was performed by evaluation of enzyme kinetics, according to Michaelis-Menten (Km- and Vmax-values), and by comparing the results with a conventional method. 17 tissue specimens (foreskin) of healthy boys (1 to 8 yrs) were examined with two methods (A and B). A: Conventional assay, using 8 pmol 3H-T as substrate. B: Evaluation of enzyme kinetics with increasing concentrations of 3H-T (8 to 208 pmol). Separation of metabolites by thin layer chromatography (TLC). Zone detection by computing radio TLC scanner. Control of purity and specific radioactivity by high resolution radio gas chromatography. Results showed age dependent values of 5&R (x̄+SEM): Vmax=18.49+2.57 pmol/mg.h; Km= 102.82+11.41 nM, with a maximum at age 5 yrs: Vmax=36.33+4.32 pmol/mg.h; Km=171.77+23.53 nM. Conclusion: It is recommended to evaluate 5&R-analysis by the enzyme kinetic method. The results of Vmax correspond to those obtained with the single point assay (method A, r=0.98). The advantage of method B is a reproduceable method giving biochemically absolute data for quality and quantitiy of the enzyme.

Distribution of [14C]aztreonam in rat tissue.

[14C]aztreonam was administered intramuscularly (50 mg/kg) to male and female rats. Groups of 10 rats (five male and five female) were sacrificed at 0.25, 2, 6, and 24 h after dosing. Blood and various tissues were removed from six rats (three male and three female) in each group for determination of total radioactivity and unchanged aztreonam by liquid scintillation counting and thin-layer radiochromatography. The remaining rats were prepared for whole-body autoradiography. Radioactivity was distributed rapidly to most tissues and was rapidly eliminated from blood into urine and bile. Concentrations of total radioactivity in kidney, liver, small and large intestines and their contents, and urinary bladder were higher than those in serum at all the times examined. Concentrations of unchanged aztreonam in serum, kidney, liver, and lung declined at about the same rate as did that of total radioactivity in the same tissues. The results of whole-body autoradiography essentially confirmed the results of the distribution of [14C]aztreonam as determined by liquid scintillation counting. No major differences between males and females were observed when concentrations in organs common to both were compared.

Pharmacologic Inhibition of Thromboxane Synthetase and Platelet Aggregation: Modulatory Role of Cyclooxygenase Products

Abstract Dazoxiben , an imidazole-derived selective inhibitor of thromboxane A2 (TxA2) synthetase, prevented TxB2 synthesis in vitro in platelet-rich plasma from 16 normal subjects. Inhibition of TxB2 synthesis was accompanied by increased generation of PGE2, PGF2 alpha, and PGD2, as shown by radioimmunoassay, thin-layer radiochromatography, and high- resolution gas chromatography-mass spectrometry. Even at dazoxiben concentrations (40–80 microM) above those inhibiting TxB2 synthesis, platelet aggregation induced by threshold concentrations of arachidonic acid was inhibited in only 4 of 16 subjects, referred to as responders. The remaining 12 individuals were defined as nonresponders. The aggregating effect of arachidonic acid and of the prostaglandin- endoperoxide analog U-46619 was potentiated by PGE2 and prevented by PGD2 at concentrations within the range of those detected in dazoxiben - treated platelet-rich plasma. The antiaggregating effect of dazoxiben was counteracted by PGE2 (in responders) and was potentiated by PGD2 (in nonresponders). Platelets from responders and nonresponders did not differ in the amount of immunoreactive PGE2 material or in their sensitivity to U-46619 or PGD2. It is concluded that inhibition of thromboxane synthetase does not per se prevent platelet aggregation. The functional result of thromboxane suppression appears to be modulated by an interplay of the prostaglandin-endoperoxides, PGE2 and PGD2, which are formed in excess.

Pharmacologic inhibition of thromboxane synthetase and platelet aggregation: modulatory role of cyclooxygenase products

Dazoxiben , an imidazole-derived selective inhibitor of thromboxane A2 (TxA2) synthetase, prevented TxB2 synthesis in vitro in platelet-rich plasma from 16 normal subjects. Inhibition of TxB2 synthesis was accompanied by increased generation of PGE2, PGF2 alpha, and PGD2, as shown by radioimmunoassay, thin-layer radiochromatography, and high-resolution gas chromatography-mass spectrometry. Even at dazoxiben concentrations (40-80 microM) above those inhibiting TxB2 synthesis, platelet aggregation induced by threshold concentrations of arachidonic acid was inhibited in only 4 of 16 subjects, referred to as responders. The remaining 12 individuals were defined as nonresponders. The aggregating effect of arachidonic acid and of the prostaglandin-endoperoxide analog U-46619 was potentiated by PGE2 and prevented by PGD2 at concentrations within the range of those detected in dazoxiben -treated platelet-rich plasma. The antiaggregating effect of dazoxiben was counteracted by PGE2 (in responders) and was potentiated by PGD2 (in nonresponders). Platelets from responders and nonresponders did not differ in the amount of immunoreactive PGE2 material or in their sensitivity to U-46619 or PGD2. It is concluded that inhibition of thromboxane synthetase does not per se prevent platelet aggregation. The functional result of thromboxane suppression appears to be modulated by an interplay of the prostaglandin-endoperoxides, PGE2 and PGD2, which are formed in excess.

The role of the kidney in the metabolism of prostacyclin by the 15-hydroxyprostaglandin dehydrogenase pathway in vivo

Five different vascular beds of the cat were tested for their ability to metabolize tritium-labeled or unlabeled prostacyclin by 15-hydroxyprostaglandin dehydrogenase in vivo. Thin-layer radiochromatography with two different solvent systems was used to characterize the radioactive metabolites formed. Following the infusion of unlabeled prostacyclin, plasma levels of 6-ketoprostaglandin F 1α and 6,15-diketo-13,14-dihydroprostaglandin F 1α immunoreactivities were monitored using previously described radioimmunoassays. No important metabolism was found in the head and the hindlimb. Plasma extracts from the vascular bed of the lung (and the heart) contained only small amounts of 6,15-diketo-13,14-dihydroprostaglandin F 1α-like material. Prostacyclin was extensively metabolized in the liver (probably by β- and ω-oxidation), but no material similar to 6,15-diketo-13,14-dihydroprostaglandin F 1α was found. In renal venous blood the predominating material showed properties similar to those of 6,15-diketo-13,14-dihydroprostaglandin F 1α with both analytical methods. In other experiments aortic blood was collected during and after infusions of tritium-labeled prostacyclin into the postcava. Two consistent peaks were demonstrated in plasma extracts of this blood, one co-chromatographing with 6-ketoprostaglandin F 1α and the other one with 6,15-diketo-13,14-dihydroprostaglandin F 1α. In nephrectomized animals the formation of the 6,15-diketo-13,14-dihydroprostaglandin F 1α-like material was not significantly impaired. Similarly, no significant differences in the formation and elimination of 6,15-diketo-13,14-dihydroprostaglandin F 1α immunoreactivity and 6-ketoprostaglandin F 1α immunoreactivity were found after nephrectomy. It is concluded that, although the kidneys have a considerable capacity to metabolize prostacyclin by 15-hydroxyprostaglandin dehydrogenase, they seem to be of little importance for the overall conversion of prostacyclin via this pathway in the total circulation.

12S,20-dihydroxyicosatetraenoic acid: a new icosanoid synthesized by neutrophils from 12S-hydroxyicosatetraenoic acid produced by thrombin- or collagen-stimulated platelets.

A new metabolite of arachidonic acid, formed during interaction between thrombin- or collagen-stimulated platelets and unstimulated neutrophils, has been demonstrated by both thin-layer radiochromatography and high-performance liquid chromatography. Production of the 3H-labeled metabolite in combined suspensions containing [3H]arachidonate-labeled platelets and unlabeled neutrophils from aspirin-treated donors suggested that platelet 3H-labeled 12S-hydroxy-5,8-cis,10-trans,14-cis-icosatetraenoic acid (12-HETE) was the precursor. This was confirmed by identification of the same product when purified 12-[3H]HETE was added directly to unstimulated neutrophils. Hydrogenation and oxidation of the isolated product, followed by gas chromatography-mass spectrometry showed the structure to be 12S,20-dihydroxyicosatetraenoic acid. These experiments further show that platelet stimuli known to occur in vivo may initiate metabolic interactions between different cell types via the arachidonic acid pathway.

An investigation of the transformations of 2-amino-2-thiazolin-4-one and its derivatives in aqueous solutions

It has been shown by thin-layer radiochromatography that the action of an aqueous solution of alkali on 2-amino-2-thiazolin-4-one and its 5-alkyl derivatives leads predominantly to the opening of the ring by a mechanism similar to the opening of lactams in an alkaline medium.

Arachidonic acid metabolism in reproductive tissues of pregnant guinea pig under in vivo circumstances

Uptake and metabolism of tritium labelled arachidonic acid have been studied under in vivo circumstances in placenta, uterus and fetal membranes of pregnant guinea pigs on days 40 and 60 of pregnancy. Distribution of radioactivity within the lipid fractions of the selected tissues showed a characteristic pattern depending on the gestational ages. Composition of labelled lipids was determined by radio thin layer chromatography and quantitated with liquid scintillation measurements. The main site of arachidonic acid incorporation was the 2-position of phosphatidylcholine. Near term, a considerable PGF 2α-synthesis from exogenously administered 3-H-arachidonic acid could be demonstrated for the first time “in vivo”.

Transport of [ 14C]coenzyme Q 10 from the liver to other tissues after intravenous administration to guinea pigs

After intravenous injection of [ 14C]coenzyme Q 10 into guinea pigs, the blood level of radioactivity declined rapidly within 30 min. However, radioactivity in the blood subsequently increased, reached a peak at 8 h after the injection and thereafter decreased slowly. Since the liver uptake of radioactivity at 30 min after the injection was 87.8% of the dose and the increase of blood level was also seen in guinea pigs with bile duct fistula, it was concluded that the radioactivity taken up by the liver was redistributed to the blood and then transferred to other tissues. Tissue radioactivity levels were highest in the liver and spleen at 30 min after the injection and decreased thereafter. The levels in the blood and kidney peaked at 8 h, while those in the heart and brain peaked at 24 h and subsequently decreased. In contrast, the level in the adrenal gland was still increasing at 168 h after the injection; it rose sharply from 30 min, and at 24 h was higher that the level in any other tissue. Electrophoretic analysis showed that the radioactivity in serum at 8 h after the injection was associated with lipoproteins. It was confirmed by thin layer radiochromatography that the radioactivities in serum and liver at various times were due to coenzyme Q 10 hydroquinone.

Release of prostaglandins from intact fetal lamb ductus arteriosus.

Intact rings and slices of ductus arteriosus from fetal lambs near term (130–143 days gestation) were incubated with [14C]arachidonic acid and the fate of the label in the incubation fluid and tissue was followed by thin-layer radiochromatography (TLC). Prostaglandins formed from endogenous stores of arachidonic acid were assayed by capillary gas chromatography with electron-capture detection. In additional experiments, the prelabelled tissue was incubated with various drugs (e.g., angiotensin II, bradykinin, and calcium ionophore) to determine their effects on the release of 14C-labelled prostaglandins. Results indicate that the ductus arteriosus is oriented towards forming prostaglandin (PG) I2, with PGE2 being formed to a minor extent in all studies. While radiochromatographic studies (TLC) suggested a considerable complexity mostly because of overlapping products (PGs and metabolites), this was resolved using gas chromatography where a complete resolution of PGs from these metabolites was observed. In the tissue-labelling experiments most of the radioactivity remained in the incubation fluid and was recovered as unchanged arachidonic acid, although a small but significant proportion of label was observed in a mixture of prostaglandins (4.2 ± 0.6%, n = 16) with 6-keto PGF1α being the major product. The ductus tissue incorporated a small amount of label (3.4 ± 0.4%, n = 19) of which 49.7 ± 4.9% (n = 13) was found in the tissue phospholipids. Hydrolysis of tissue-bound [14C]arachidonic acid, which appeared in the perfusing medium together with 14C-labelled prostaglandins, was in the range of 28.0 ± 2.7% (n = 9) of total tissue radioactivity per experiment. Although 42.2 ± 2.5% (n = 18) of the radioactivity released by each of the drugs represented a mixture of prostaglandins and their metabolites (the rest recovered as arachidonic acid), no appreciable selectivity by any of the drugs was found for either the stimulation of phospholipase activity in this tissue or for the rerouting of the prostaglandin synthetic system to form prostaglandin E2 instead of I2. We conclude that prostaglandin I2 rather than E2 is probably of physiological importance in this unique fetal blood vessel.

Vessel wall arachidonate metabolism after angioplasty: Possible mediators of postangioplasty vasospasm

The mechanism of postangioplasty vasospasm is uncertain. It was postulated that a reduction in vasodilator prostaglandin I2 (PGI2) or prostaglandin E2 (PGE2) or an increase in vasoconstrictor hydroperoxy acids might contribute to spasm of a dilated artery. Twelve mongrel dogs were anesthetized, intubated, and mechanically ventilated. Heart rate and aortic pressures were continuously monitored and arterial blood gases maintained within physiologic limits. A single carotid artery was dilated in each animal (4 atm × 1 minute × 3) using nonex-pandable polyethylene balloon catheters with inflated balloon diameters 50 to 100% larger than the internal arterial lumen. The opposite carotid artery served as a control. In 4 animals, aspirin (10 mg/kg, intravenously) was injected 30 minutes before dilation. Sixty minutes after dilation, animals were heparinized and the carotid arteries carefully removed. The in vitro conversion of carbon-14(14C) -arachidonic acid (AA) to 6-keto PGF1α (PGI2), PGE2, and 12L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) was determined using thin-layer radiochromatography. Angioplasty caused a 70% decrease in vessel wall PGI2 production and a 44% decrease in PGE2 production (n = 4, p < 0.05). Reductions in in vitro conversion of 14C-AA to PGI2 and PGE2 induced by angioplasty were comparable to that produced by cyclooxygenase inhibition with aspirin. Angioplasty, in addition, caused a 104% increase in vessel wall HETE production (n = 4, p < 0.05). Therefore, angioplasty results in a local derangement of AA metabolism characterized by decreases in vasodilator prostaglandins and increases in vasoconstrictor hydroperoxy acids. These local changes may contribute, in part, to sudden arterial occlusion after angioplasty.

Biodegradation of a cationic surfactant in activated sludge

The biodegradation of DTDMAC (ditallowdimethylammonium chloride, a fabric softening agent) was established in semi-batch activated sludge reactors. Three 14C-forms of DTDMAC were studied separately under the simulated organic loading rates of conventional and extended aeration activated sludge treatment. Primary biodegradation was shown by means of a specific analytical technique in combination with radiochemical procedures. Ultimate biodegradation for each carbon position in the DTDMAC molecule was established by the detection of 14CO 2. Intermediate metabolites were followed throughout the study by both radio thin-layer chromatography and radiochemical procedures. Each carbon position of the DTDMAC molecule was equally accessible to metabolism and ultimate degradation by acclimated microorganisms. Degradation occurred more rapidly under extended aeration conditions and was influenced by the strong tendency of DTDMAC to associate with the microbial population. The low levels of metabolites observed were not of a single classification or characteristic and they did not persist in the activated sludge. This study suggests that DTDMAC removal in an activated sludge plant is a result of both sorption/precipitation and biodegradation mechanisms.

PGD 2 formation in the vasculature: Characteristics of rat tail vein prostaglandin endorperoxide-D isomerase

Rat tail vein homogenates, microsome and high speed supernatant fractions were incubated with [1- 14C]prostaglandin endoperoxide (PGH 2) and products separated and identified by radio-thinlayer chromatography. PGI 2 synthase was localized to the microsomal fraction, but exhibited low activity compared to rat tail arteries prepared in the same manner. PGH-D isomerase was maximally active in the presence of reduced glutathione at pH 7.5–8.0, exhibited a K m for PGH 2 of 33 μM, and was inhibited sulfhydryl-directed reagents. The similarities of this enzyme to PGD synthase of the rat cerebral microvasculature are discussed.

AD 6, a coronary dilating agent, stimulates PGI2 production in rat aorta ex vivo and in human endothelial cells in culture

Summary AD6(8-monochloro-3-beta-diethylaminoethyl-4-methyl-7-ethoxy-carbonyl-methoxy-coumarin) reportedly exerts specific coronary vasodilatory activity and reduces platelet aggregation in laboratory animals. We investigated whether AD6 stimulates prostacyclin (PGI 2 ) synthesis by vascular cells. AD6 treatment of rats induced a dose dependent enhancement of prostacyclin (PGI2) synthesis by rings of thoracic aorta measured by bioassay and confirmed by thin layer radio-chromatography of the products of 14 C arachidonic acid( 14 CAA) metabolism. In vitro incubation of the drug with cultured human endothelial cells caused a concentration dependent stimulation of PGI2 production measured by radioimmunoassay of 6keto PGF1 α . It is proposed that the previously described vasodilatory activity of the drug might be mediated through the stimulation of PGI2 release.

Human neutrophils transform prostaglandins by a myeloperoxidase-dependent mechanism.

Intact human neutrophils, incubated with the soluble stimulant phorbol myristate acetate, discharge lysosomal components, generate oxygen metabolites, and transform exogenous 6-keto-prostaglandin F1 alpha, prostaglandin E2, and prostaglandin F2 alpha as assessed by thin layer radiochromatography. Neutrophils alone were incapable of transforming the prostaglandins. The addition of catalase or the myeloperoxidase inhibitor, azide, protected all three prostaglandins from the phorbol-stimulated neutrophils. Neither superoxide dismutase, heat-inactivated catalase, nor albumin had any inhibitory effect in this system. A model system consisting of glucose-glucose oxidase, as a source of H2O2, purified myeloperoxidase, and chloride was also able to transform the prostaglandins in an identical fashion. Neither glucose-glucose oxidase alone nor glucose-glucose oxidase and myeloperoxidase under chloride-free conditions were able to mediate this transformation. Thus, it appears that intact human neutrophils can transform prostaglandins by a mechanism dependent on H2O2, the lysosomal enzyme myeloperoxidase, and chloride. Given the importance of prostaglandins in regulating immune function, neutrophil-dependent prostaglandin transformation could play a novel role in modulating the inflammatory response.

Mitochondrial hydroxylation of the cyclohexane ring as a result of beta-oxidation blockade of a cyclohexyl substituted fatty acid.

Among the urinary metabolites of dodecylcyclohexane or cyclohexylacetic acid, the glycine conjugate of 1-hydroxy-cyclohexylacetic acid was identified and its origin studied, using cyclohexylacetic acid as the starting molecule, as it results from beta-oxidation of cyclohexyldodecanoic acid produced by terminal oxidation of the alkyl chain of the cycloparaffin. Three hypotheses were tested: (a) hydroxylation by the liver microsomal mixed-function oxidases involved in detoxication mechanisms; (b) hydroxylation by a cyt. P450-containing mitochondrial hydroxylase; and (c) beta-oxidation blockade after the reaction catalyzed by enoyl-CoA-hydratase. Liver microsomal or mitochondrial fractions were prepared and incubated in the presence of [14C] cyclohexylacetic acid, glucose-6-phosphate dehydrogenase and a NADPH-producing system. On the other hand, mitochondria were incubated in a suitable respiratory medium with or without cofactors required for ATP production. The reaction products were extracted and analyzed by thin layer radiochromatography and radio gas chromatography. Evidence is given that hydroxylation of cyclohexylacetic acid in position 1 is a mitochondrial step requiring activation in the acyl-CoA form and results from beta-oxidation blockade, the cyclohexane ring hindering hydroxyacyl-CoA-dehydrogenase action.

Caffeine metabolism in liver slices during postnatal development in the rat

The metabolism of caffeine was investigated in liver slices of young and adult rats. Liver slices from adult male rats metabolized caffeine at an initial rate of 48.31 ± 3.71 nmoles · (g liver) −1 · hr −1 to four main metabolite fractions. By a combination of thin-layer radiochromatography and high performance liquid chromatography, theophylline, paraxanthine and 1, 3, 7-trimethyldihydrouric acid were identified as caffeine metabolites. Apparent V max of the overall reaction was 83.30 nmoles caffeine metabolites formed · (g liver) −1 · hr −1. Theophylline competitively inhibited caffeine metabolism [the apparent K m was 19.20, μM in the absence of theophylline, the apparent k i was 36.50 μM in the presence of theophylline (100 μM)]. SKF 525-A inhibited caffeine metabolism; the formation of all of the metabolite fractions was inhibited to a similar extent. Allopurinol (100 μM) had no effect. The specific activity of the enzyme system was extremely low when liver slices of 2-day-old-rats were used [1.46 ± 0.08 nmoles caffeine metabolites formed · (g liver) −1 · hr −1]; the reaction velocity increased gradually with increasing age and reached a peak [52.26 ± 1.41 nmoles caffeine metabolites formed · (g liver) −1 · hr −1]at 30 days of age. Changes in the formation of the four metabolite fractions with age followed the pattern of the overall caffeine metabolism. These results demonstrate that the liver of the newborn rat has an extremely limited capacity to metabolize caffeine in vitro and are consistent with the proposed involvement of the liver microsomal cytochromes P-450 monooxygenase system in the metabolism of caffeine. N-Demethylation is the main pathway of in vitro caffeine metabolism in the rat liver at all ages.

Evaluation of prostaglandin biosynthetic activity in canine basilar artery following subarachnoid injection of blood.

Transformation of arachidonic acid into prostaglandins was investigated in the basilar artery by incubating sections of artery with carbon-14-labeled arachidonic acid. Thin-layer radiochromatography revealed that, in normal canine basilar arteries, 14C-arachidonic acid was transformed mainly to 6-keto-prostaglandin (PG)F1 alpha, a spontaneous metabolite of prostacyclin (PGI2). Among other prostaglandins, only a small amount of PGF2 alpha was detected, whereas PGD2, PGE2, and thromboxane B2 were not. Arteries removed on Days 3 and 8 after subarachnoid blood injection showed a prostaglandin synthesis profile similar to that in the normal cerebral artery. In borate-buffered saline (0.1M borate buffer, pH 9.0/0.15M NaCl = 1:9, vol/vol), canine basilar artery produced a PGI2-like substance that inhibited adenosine diphosphate (ADP)-induced platelet aggregation. Its anti-aggregatory activity was completely abolished by acidification. Aspirin likewise inhibited production of the anti-aggregatory substance. From these results, it was concluded that the anti-aggregatory activity was due solely to the production of PGI2 by the arterial specimen. Based on the above results, PGI2 biosynthetic activity in the cerebral artery exposed to subarachnoid blood injection was bioassayed by measuring the inhibitory activity of the incubation product upon ADP-induced platelet aggregation following incubation of the arteries in borate-buffered saline for 5 to 30 minutes at 20 degrees C, using synthetic PGI2-Na as a standard. The synthetic activity of PGI2 in the artery exposed to subarachnoid blood injection had diminished remarkably by Days 3 and 8. This diminution of PGI2 synthesis in the cerebral artery may be involved in the pathogenesis of cerebral vasospasm.

Thromboxane synthesis in megakaryocytes isolated by centrifugal elutriation

A systematic approach to the optimization of centrifugal elutriation is described for the purification of rat femoral marrow megakaryocytes, which has permitted the first direct demonstration of thromboxane synthesis in these cells. The rapidity, simplicity, and capacity of this technique has made it possible to process 2 x 10(9) marrow cells in the absence of platelet aggregation inhibitors in 20 min to obtain 10(6) megakaryocytes at a recovery of about 80% with a concentration of up to 282-fold. Incubation of these isolated megakaryocytes with 14C-arachidonic acid and subsequent thin-layer radiochromatography has revealed thromboxane B2 as the major cyclooxygenase pathway metabolite of arachidonic acid in megakaryocytes.