Metabolism Of 3H Research Articles

The Metabolism of 3H and 14C with Special Reference to Radiation Protection

The Metabolism of 3H and 14C with Special Reference to Radiation Protection

Metabolism of 3H- and 14C-labeled glutamate, proline, and alanine in normal and adrenalectomized rats using different sites of tracer administration and sampling

Alanine, glutamate and proline labeled with 14C and 3H were infused into fasted normal and adrenalectomized rats. Alanine was administered by the A-V mode (arterial administration-venous sampling), and glutamate and proline by both the A-V and V-A (venous administration-arterial sampling) modes. The kinetics of 14C alanine and 14C glutamate differed markedly from those of the tritium-labeled compounds, but there was little difference in the kinetics of 3H and 14C proline. The replacement rate calculated from the A-V mode for glutamate was about half that obtained in the V-A mode, but there was little difference with proline. The masses of the amino acids (total content of amino acids in the body) were calculated from the washout curves of the tritium-labeled compounds after the infusion of tracer was terminated. The masses for the normal rats were 407 μmol/kg for alanine, 578 μmol/kg for glutamate and 296 μmol/kg for proline. The so-called distribution spaces calculated conventionally from total masses and the amino acid concentrations in plasma are much greater than the volume of the body, reflecting the fact that amino acid concentrations in tissues greatly exceed those in plasma. Adrenalectomy markedly affected the kinetics of the three amino acids, and their replacement rates were greatly reduced. The proline and glutamate masses were reduced by at least one half, while that of alanine was unchanged. Adrenalectomy markedly reduced the conversion of proline to glutamate. The hydrocortisone regimen used in this study restored the metabolism of alanine and glutamate to normal, but had no effect on that of proline.

Uptake and metabolism of 3H-(+/-)-noradrenaline in the isolated perfused rat liver.

The influence of inhibitors of metabolism and uptake of noradrenaline on the 3H-noradrenaline removal from the perfusion fluid by the isolated rat liver was studied. Livers were perfused with 3 nmol/l 3H-noradrenaline and 3H-noradrenaline and 3H-metabolites were determined in effluent, liver and bile. After the perfusion with 14,900 +/- 920 dpm.g-1.min-1 during 90 min, cumulative removal of tritium was 323,574 +/- 63,103 dpm/g. 3H-metabolites recovered from the liver after 90 min perfusion represented 71.1 +/- 9.0% of total metabolite formation. Only the OMDA-fraction appeared in the perfusate; its approach to steady state of efflux was slow. The inhibition either of MAO or COMT changed neither the total removal of tritium nor the 3H-metabolites recovered from the liver. Cocaine (10 mumol/l) reduced the accumulation of 3H-noradrenaline in the liver. The uptake2 inhibitor corticosterone (30 mumol/l) diminished total removal of tritium and the 3H-metabolites recovered from the liver without changing the accumulation of 3H-noradrenaline. The hypothesis of two different compartments, one responsible for the metabolism and the other for the accumulation of the amine is discussed.

Kinetic constants for uptake and metabolism of 3H-(-)noradrenaline in rabbit aorta. Possible falsification of the constants by diffusion barriers within the vessel wall.

1. The neuronal uptake of 3H-(-)noradrenaline into aortic rings from reserpine-pretreated animals was a saturable process with a Km of 2.3 mumol X l-1 and a Vmax of 0.5 nmol X g-1 X min-1. Similar constants were obtained when the neuronal deamination of noradrenaline was taken as an index for neuronal uptake. When the tissue was incubated in the usual way, i.e., when the amine was allowed to enter the aortic rings via both the intimal and the adventitial surface, then there was no initial delay (tlag) for the neuronal uptake of noradrenaline (MAO and COMT inhibited). On the other hand, when the amine entry was restricted to the intimal surface, there was a tlag of 2 min, probably due to the slow equilibration of the extracellular space of the media with the incubation medium. Furthermore, for low amine concentrations the rate of uptake in the latter situation was about 10 times lower than that in the former one. Thus, the rate of uptake clearly depended on the way the amine entered the tissue. 2. The corticosterone-sensitive extraneural uptake of 3H-(-)noradrenaline determined in nerve-free aortic rings was characterised by a high Km (490 mumol X l-1) and Vmax (35 nmol X g-1 X min-1). For uptake2 a tlag of 1 min was observed. 3. The analysis of the extraneuronal O-methylating system in nerve-free aortic rings yielded a Km of 3.6 mumol X l-1 and a Vmax of 0.6 nmol X g-1 X min-1. The tlag for the O-methylation of noradrenaline was in the same order of magnitude as that for uptake2. At low amine concentrations the corticosterone-sensitive accumulation of noradrenaline was prevented by COMT, but not by MAO. The latter enzyme reduced the steady-state accumulation of noradrenaline by about 50%. When the amine entry was restricted to one surface only, the results indicated that the extraneuronal O-methylation of noradrenaline generated a steep concentration gradient of the parent amine within the extracellular space of the aorta. 4. All saturable processes fitted the Michaelis-Menten equation. However, kinetic constants determined in incubated organs may be falsified by poor diffusion of the substrate through the extracellular space.

Metabolism of 3H- and 14C-labelled lactate in starved rats

Metabolism of 3H- and 14C-labelled lactate in starved rats

Kinetics of the uptake and metabolism of 3H-(�) isoprenaline in the rat submaxillary gland

1. The uptake and O-methylation of 3H-(±)isoprenaline was studied in slices of the rat submaxillary gland. 2. The initial uptake of 3H-isoprenaline after inhibition of catechol-O-methyl transferase (COMT) was described by a single saturable process with relatively high Km (311 μM) and Vmax (101 nmoles·g−1·min−1). Both corticosterone and normetanephrine were competitive inhibitors of uptake. 3. When examined at substrate concentrations lower than the Km for uptake (and after block of COMT), 3H-isoprenaline distributed into two compartments in the tissue which approached equilibrium with half times of 2.4 and 15.8 min. The filling of both compartments was inhibited by corticosterone or phenoxybenzamine and also by high-K+ medium (in which 118 mM NaCl of the incubation medium had been replaced by KCl), but remained unaffected on substituting 118 mM NaCl with Tris-HCl. 4. In tissues in which COMT was not inhibited, the metabolism of 3H-isoprenaline to 3H-O-methylisoprenaline proceeded at a constant rate from the beginning of the incubation with the amine. When the substrate concentration was very low, little unchanged 3H-isoprenaline was found in the tissue. On the other hand, at high substrate concentrations the parent amine accumulated in the tissue, and at a time when 0-methylation had reached a steady state, the accumulation of 3H-isoprenaline was continuing. 5. The formation of 3H-O-methylisoprenaline was impaired by the presence of corticosterone, normetanephrine, phenoxybenzamine or 17-β-oestradiol with no indication of inhibition of COMT. While lowering the external Na+ concentration (on replacing 118 mM NaCl by 236 mM sucrose) did not affect the formation of 3H-O-methylisoprenaline, replacement of 118 mM NaCl by KCl reduced it. 6. The dependence of the steady-state rate of formation of 3H-O-methylisoprenaline on the substrate concentration in the incubation medium showed that two saturable components participated in the O-methylation of 3H-isoprenaline (low Km system: Km=7.2 μM and Vmax=1.2 nmoles·g−1·min−1; high-Km system: Km=339 μM and Vmax=4.6 nmoles·g−1·min−1). Corticosterone and normetanephrine competitively inhibited both the low-Km and the high-Km O-methylation. 7. The results indicate that the submaxillary gland of the rat resembles other tissues in having a low-Km (high-affinity) “O-methylating system” as well as a high-Km (low-affinity) extraneuronal uptake mechanism for catecholamines.

The kinetic constants for the extraneuronal uptake and metabolism of 3H-(-)-noradrenaline in the perfused rat heart.

1. Rat hearts were perfused with 50–2000 μM 3H-(−)-noradrenaline in the presence of 14C-sorbitol (to measure the extracellular distribution of the amine) and of 100 μM cocaine (to inhibit neuronal uptake). Initial rates of removal of noradrenaline were determined. Extraneuronal uptake determined in this way consisted of two components: a saturable and a non-saturable one. 2. When monoamine oxidase (MAO) was inhibited, the kinetic constants for saturable extraneuronal uptake of noradrenaline were: Km 60 μM, Vmax 50 nmoles · g−1 · min−1. U-0521, an inhibitor of catechol-O-methyl transferase (COMT) acted like a competitive inhibitor of extraneuronal uptake: the Km increased, Vmax remained unaffected. 3. In separate experiments either MAO or COMT was intact. Rat hearts were perfused with various concentrations of 3H-(−)-noradrenaline until the metabolites appeared at a constant rate in the venous effluent. These steady-state rates of metabolite formation were used to determine the kinetic parameters of the metabolizing systems. 4. O-methylation of noradrenaline in the intact heart was a saturable process and obeyed Michaelis-Menten kinetics; it had very low Km (1.7 μM) and Vmax (1.2 nmoles · g−1 · min−1). 5. Also the deamination of noradrenaline by the perfused heart was a saturable process, but Km and Vmax were considerably higher than for O-methylation (140 μM and 25 nmoles · g−1 · min−1, respectively). 6. It is concluded that “extraneuronal uptake and subsequent metabolism” of noradrenaline represents a site of loss that is highly effective at very low substrate concentrations (i.e., below the Km of the two metabolizing systems).

The neuronal and extraneuronal uptake and metabolism of 3H-(-)-noradrenaline in the perfused rat heart.

1. Hearts were obtained from reserpine-pretreated rats and perfused with 0.95 micron 3H(-)-noradrenaline. The rate of removal of 3H-noradrenaline from the perfusion fluid was measured (from the arterio-venous difference) as well as the rate at which the 3H-metabolites appeared in the venous effluent. 2. When either 30micron corticosterone was added under steady-state conditions during perfusion with 3H-noradrenaline (to inhibit neuronal and extraneuronal uptake, respectively), each inhibitor reduced the removal of noradrenaline by about 50%; in the presence of both inhibitors removal was abolished. 3. Dihydroxymandelic acid (DOMA) was of neuronal, normetanephrine (NMN) of extraneuronal origin; dihydroxyphenylglycol (DOPEG) and the OMDA fraction (containing methoxyhydroxyphenylglycol-MOPEG-and methoxyhydroxymandelic acid-VMA) were formed both neuronally and extra-neuronally. 4. The extraneuronal metabolism of 3H-noradrenaline was in quick equilibrium with the 3H-noradrenaline in the perfusion fluid; most of the total formation of DOPEG, MOPEG and NMN was recovered from the venous effluent. 5. Extraneuronally formed DOPEG, MOPEG and NMN distributed in the tissue with half times corresponding to their half time for efflux. 6. Inhibition of monoamine oxidase (MAO) by pargyline increased the extraneuronal formation of NMN; MAO and catechol-O-methyl transferase (COMT) appear to be contained in the same extraneuronal compartment. 7. The extraneuronal accumulation of 3H-noradrenaline required 30 min or more to reach a steady state; inhibition of one or both enzymes slowed this process. Inhibition of MAO increased the extra-neuronal accumulation of 3H-noradrenaline; inhibition of COMT failed to do so, since the enzyme inhibitor (U-0521) was a weak inhibitor of extra-neuronal uptake. 8. The rate constants for the efflux of the metabolites of noradrenaline decreased in the order of MOPEG greater than DOPEG greater than NMN greater than DOMA greater than VMA.

The effects of labetalol (AH 5158) on metabolism of 3H(−)-noradrenaline released from the cat spleen by nerve stimulation

The competitive α and β adrenoceptor antagonist labetalol, in concentrations up to 10 −4 M, produced a dose dependent increase in overflow of 3H and [ 3H]noradrenaline in the isolated blood perfused cat spleen following stimulation of the splenic nerves at a frequency of 10 Hz. Labetalol had no effect on the pattern of overflow of label following stimulation. In experiments in which the metabolism of [ 3H]noradrenaline released on nerve stimulation was examined, labetalol produced a concentration dependent increase in the percentage of [ 3H]noradrenaline and a decrease in the percentage of [ 3H]DOPEG in the venous blood following nerve stimulation. Production of [ 3H]COMT metabolites and [ 3H]DOMA was not affected. It is suggested that in the isolated blood perfused cat spleen labetalol produces the elevation of overflow and effects on noradrenaline metabolism by inhibition of neuronal uptake of noradrenaline. The drug has no detectable effects on the enzymes MAO or COMT or on extraneuronal uptake.

Relationship of aryl hydrocarbon hydroxylase activity to benzo(a)pyrene-metabolizing activity of cells in culture

The basal level of aryl hydrocarbon hydroxylase (AHH) activity was high in cultures of low passage hamster embryo (HE) cells but AHH inducibility by benz(a)-anthracene (BA) was low; in R72/3 rat liver cells, basal activity was low and inducibility was high. The metabolism of 3H--benzo(a)pyrene was similar in cultures of BA-induced R72/3 cells and uninduced HE cells. Thus, low AHH inducibility may not always be an indication of the cells' absolute hydrocarbonmetabolizing capacity.

Stereoselectivity in the metabolism of 3H-noradrenaline during uptake into and efflux from the isolated rat vas deferens.

1. The metabolism of 3H-(-)- and 3H-(±)-noradrenaline (NA) was studied in the isolated rat vas deferens either under conditions of uptake or of efflux of the amine. Any differences obtained between 3H-(-)-and 3H-(±)NA as substrate were interpreted as being a reflection of differences between the two isomers of the amine. 2. Uptake experiments (0.13 μM; 7.5 min) showed that neuronal mechanisms of amine disposition prevail over extraneuronal ones. Thus, most of the metabolites of 3H-NA formed during incubation with the amine (including the O-methylated products) were of neuronal origin. The acid deaminated metabolite 3,4-dihydroxymandelic acid (DOMA), tended to be much better retained by the tissue than the neutral deaminated metabolite, 3,4-dihydroxyphenylethyleneglycol (DOPEG). While neuronal uptake exhibited no stereoselectivity, a pronounced stereoselectivity was found for monoamine oxidase (MAO) [(-)NA> (+)NA] as well as for the enzymes which are in series with MAO, namely, aldehyde reductase and aldehyde dehydrogenase [(-)DOPEG> (+)DOPEG; (-)DOMA <(+)DOMA]. 3. After about 2 h of washout, the efflux of radioactivity from the tissue [which was previously incubated for 30 min with 1.2 μM of either 3H-(-)- or 3H-(±)NA] originated from one neuronal compartment with no stereoselectivity of the rate constant for the efflux of total tritium. The rate-limiting step for the neuronal efflux of tritium resided either in the net efflux of amine from the storage vesicles (normal tissues) or in the net efflux across the axonal membrane (tissues with the amine metabolizing enzymes inhibited). The effects of cocaine and phenoxybenzamine on the neuronal efflux of tritiated compounds strongly depended on the intraneuronal distribution of the 3H-amine. The results indicate that cocaine has only one site of action (neuronal uptake), while phenoxybenzamine exerts reserpine-like as well as cocaine-like effects. 4. The neuronal efflux of tritium from normal tissues preloaded with 3H-(-)- or 3H-(±)NA consisted mainly of amine metabolites (90% of the total; most of this was DOPEG). Since after 2 h of washout the tissue contained hardly any metabolites, these metabolites did not represent pre-formed metabolites (formed during the period of preloading) but newly formed metabolites resulting from the catabolism of the neuronally stored amine. This catabolism was brought about through the activity of presynaptic enzymes and was stereoselective in that more DOPEG, less DOMA and less O-methylated metabolites were formed from (-)-than from (+)NA.

Conjugation of l-DOPA and its metabolites after oral and intravenous administration to Parkinsonian patients

Tracer quantities of 3H- l-dopa were given either orally or intravenously to patients with Parkinson's disease. The metabolism of 3H- l-dopa was estimated by measurement of 3H- l-dopa and some of its metabolites in urine samples collected during the first 24 hr. The decarboxylation of 3H- l-dopa to 3H-dopamine was approximately the same when 3H- l-dopa was given by the two routes. The subsequent metabolism of the newly formed 3H-dopamine was markedly dependent upon the route of administration. Conjugation was the major metabolic route of 3H-dopamine derived from 3H- l-dopa given orally, while O-methylation and deamination of 3H-dopamine to 3H-homovanillic acid predominated when 3H- l-dopa was given intravenously. In contrast to the metabolism of tracer quantities of oral 3H- l-dopa, large oral doses of l-dopa 3·0 g/day) resulted in a smaller proportion of conjugated dopamine and a greater proportion of homovanillic acid. The conjugation of 3H-dopamine after tracer quantities of intravenous 3H- l-dopa was only slightly decreased by the concomitant administration of 3·0 g/day of oral l-dopa. There is evidence that conjugation occurs primarily in the gastrointestinal-hepatic system after oral administration of l-dopa. When conjugation is either suppressed by large quantities of oral l-dopa or avoided by intravenous l-dopa, the amounts of free dopamine as well as homovanillic acid are increased. Thus, under these conditions, more free dopamine is available to produce the peripheral side effects of l-dopa therapy.

Metabolism of 3H- l-dopa by the rat gut in vivo—evidence for glucuronide conjugation

The metabolism of intravenous 3H- l-dopa by the rat gut in vivo has been studied. After a single i.v. bolus of a pharmacologic dose of l-dopa, the rat duodenum accumulated 3H-noncatechol metabolites of dopa at a far greater rate than stomach, spleen or heart. 3H-noncatechols were localized predominantly in the duodenal mucosa and not the muscularis. In the mucosa, 3H-noncatechols accounted for 90 per cent of the total 3H, most of which was in the amino acid noncatechol fraction after Chromatographic separation on Alumina and Dowex. Accumulation of noncatechol metabolites in duodenal mucosa was not dependent on the administration of a pharmacologic dose of dopa; after a tracer dose of 3- l-dopa the per cent of 3H-noncatechols in the amino acid fraction actually increased. The pattern of dopa metabolites in duodenal mucosa was substantially different from a variety of other tissues, but was similar to liver, jejunum and colon. Diversion of the bile by cannulation of the common bile duct prior to the administration of 3H- l-dopa did not change the accumulation or pattern of metabolites in the duodenum. Analysis of the noncatechol amino acid fraction from duodenal mucosa by incubation with Glusulase and β-glucuronidase, thin-layer chromatography, and fluorescent assay revealed that the compounds accumulated by duodenal mucosa were largely glucuronide conjugates of catechols, particularly dopamine. It is concluded that circulating dopa is taken up by the rat intestinal mucosa, decarboxylated, conjugated and stored, largely in the form of glucuronide conjugates. The gut thus makes a major contribution to the over-all metabolism of circulating dopa. Dopa metabolites stored in the gut, moreover, are a potential reservoir of catechols for reutilization by the organism, and this may have important implications for the clinical pharmacology of l-dopa in man.