In vitro oxidation of [U-14C] palmitate, [1-14C] and [6-14C] glucose in newborn and adult rats and pigs

Studies have been made on the intensity of oxidation of [U-14C]-palmitate, [1-14C]- and [6-14C]-glucose by slices of the liver and skeletal muscles of new-born, 1-day, 5-day and adult Wistar rats and domestic pigs. It was found that the level of 14CO2 production from these substrates is higher in tissues of rats than in those of pigs. At early stages of ontogenesis, in tissues of both species intensive oxidation of glucose is observed together with oxidation of fatty acids. In the course of ontogenetic development, the intensity of glucose utilization significantly decreases, whereas the level of fatty acid catabolism remains relatively unaffected.     

Hormonal control of [14C]glucose synthesis from [U-14C]dihydroxyacetone and glycerol in isolated rat hepatocytes.

The hormonal control of [14C]glucose synthesis from [U-14C-A1dihydroxyacetone was studied in hepatocytes from fed and starved rats. In cells from fed rats, glucagon lowered the concentration of substrate giving half-half-maximal rates of incorporation while it had little or no effect on the maximal rate. Inhibitors of gluconeogenesis from pyruvate had no effect on the ability of the hormone to stimulate the synthesis of [14C]glucose from dihydroxyacetone. The concentrations of glucagon and epinephrine giving half-maximal stimulation from dihydroxacetone were 0.3 to 0.4 mM and 0.3 to 0.5 muM, respectively. The meaximal catecholamine stimulation was much less than the maximal stimulation by glucagon and was mediated largely by the alpha receptor. Insulin had no effect on the basal rate of [14C]clucose synthesis but inhibited the effect of submaximal concentration of glucagon or of any concentration of catecholamine. Glucagon had no effect on the uptake of dihydroxyacetone but suppressed its conversion to lactate and pyruvate. This suppression accounted for most of the increase in glucose synthesis. In cells from gasted rats, where lactate production is greatly reduced and the rate of glucose synthesis is elevated, glucagon did not stimulate gluconeogenesis from dihydroxyacetone. Findings with glycerol as substrate were similar to those with dihyroxyacetone. Ethanol also stimulated glucose production from dihydroxyacetone while reducing proportionately the production of lactate. Ethanol is known to generate reducing equivalents fro clyceraldehyde-3-phosphate dehydrogenase and presumably thereby inhibits carbon flux to lactate at this site. Its effect was additive with that of glucagon. Estimates of the steady state levels of intermediary metabolites and flux rates suggested that glucagon activated conversion of fructose diphosphate to fructose 6-phosphate and suppressed conversion of phosphoenolpyruvate to pyruvate. More direct evidence for an inhibition of pyruvate kinase was the observation that brief exposure of cells to glucagon caused up to 70% inhibition of the enzyme activity in homogenates of these cells. The inhibition was not seen when the enzyme was assayed with 20 muM fructose diphosphate. The effect of glucagon to lower fructose diphosphate levels in intact cells may promote the inhibition of pyruvate kinase. The inhibition of pyruvate kinase may reduce recycling in the pathway of gluconeogenesis from major physiological substrates and probably accounts fromsome but not all the stimulatory effect of glucagon.     

Comparative utilization in vivo of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose and [1-C14] palmitate in the rat

Female rats were injected i.v. with comparable trace amounts of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose, or [1-14C] palmitate, and killed 30 min afterwards. The radioactivity remaining in plasma at that time was maximal in animals receiving [U-14C] glucose while the appearance of radioactive lipids was higher in the [U-14C] glycerol animals than in other groups receiving hydrosoluble substrates. The carcass, more than the liver, was the tissue where the greatest proportion of radioactivity was recovered, while the greatest percentage of radioactivity appeared in the liver in the form of lipids. The values of total radioactivity found in different tissues were very similar when using either labelled glucose or glycerol but the amount recovered as lipids was much greater in the latter. The maximal proportion of radioactive lipids appeared in the fatty-acid form in the liver, carcass, and lumbar fat pads when using [U-14C] glycerol as a hydrosoluble substrate, and the highest lipidic fraction appeared in adipose tissue as labelled, esterified fatty acids. In the spleen, heart, and kidney, most of the lipidic radioactivity from any of the hydrosoluble substrates appeared as glyceride glycerol. The highest proportion of radioactivity from [1-14C] palmitate appeared in the esterified fatty acid in adipose tissue, being followed in decreasing proportion by the heart, carcass, liver, kidney, and spleen. Thus at least in part, both labelled glucose and glycerol are used throughout different routes for their conversion in vivo to lipids. A certain proportion of glycerol is directly utilized by adipose tissue. The fatty acids esterification ability differs among the tissues and does not correspond directly with the reported activities of glycerokinase, suggesting that the alpha-glycerophosphate for esterification comes mainly from glucose and not from glycerol.     

Enzymatic synthesis of [U-14C] ribose-labeled inosine.

A very potent anticholinesterase compound, 7-(diethoxyphosphinyloxy)-N-methylquinolinium fluorosulfate, has been used to determine the normality of acetylcholinesterase solutions. The inhibitor reacts rapidly and completely with acetylcholinesterase. The bimolecular rate constant is 2.5 × 10⁸m^?1 min^?1 and the equilibrium constant is about 10⁶. The reaction produces an inactive diethylphosphoryl enzyme in which the active serine is phosphorylated. The reaction produces the highly fluorescent 1-methyl-7-hydroxyquinolinium dipolar ion as a leaving group. The inhibited enzyme is quite stable and hydrolyzes to produce active enzyme only at the rate of 0.04%/min. The inhibitor was used in two ways for measuring the normality of acetylcholinesterase solutions: (1) The very fast reaction of the inhibitor with cholinesterase makes it convenient to determine the normality of enzyme solutions by measuring the decrease in enzyme activity caused by the addition of an accurately known quantity of the inhibitor. (2) The highly fluorescent nature of the leaving group makes it possible to measure the low concentration that is produced by the reaction of excess inhibitor with the enzyme. The two methods yielded activities per site of 6.9 × 10⁵ min^?1 and 7.3 × 10⁵ min^?1 using enzyme normalities of 1–2 × 10^?8m and 1–5 × 10^?m, respectively, using a commercial 11 S enzyme preparation from electric eel and acetylthiocholine as the enzyme substrate.     

Measurement of exogenous carbohydrate oxidation: a comparison of [U-14C]glucose and [U-13C]glucose tracers

The purpose of this study was to assess the level of agreement between two techniques commonly used to measure exogenous carbohydrate oxidation (CHO(EXO)). To accomplish this, seven healthy male subjects (24 +/- 3 yr, 74.8 +/- 2.1 kg, V(O2(max)) 62 +/- 4 ml x kg(-1) x min(-1)) exercised at 50% of their peak power for 120 min on two occasions. During these exercise bouts, subjects ingested a solution containing either 144 g glucose (8.7% wt/vol glucose) or water. The glucose solution contained trace amounts of both [U-13C]glucose and [U-14C]glucose to allow CHO(EXO) to be quantified simultaneously. The water trial was used to correct for background 13C enrichment. 13C appearance in the expired air was measured using isotope ratio mass spectrometry, whereas 14C appearance was quantified by trapping expired CO(2) in solution (using hyamine hydroxide) and adding a scintillator before counting radioactivity. CHO(EXO) measured with [13C]glucose ([13C]CHO(EXO)) was significantly greater than CHO(EXO) measured with [14C]glucose ([14C]CHO(EXO)) from 30 to 120 min. There was a 15 +/- 4% difference between [13C]CHO(EXO) and [14C]CHO(EXO) such that the absolute difference increased with the magnitude of CHO(EXO). Further investigations suggest that the difference is not because of losses of CO2 from the trapping solution before counting or an underestimation of the "strength" of the trapping solution. Previous research suggests that the degree of isotopic fractionation is small (S. C. Kalhan, S. M. Savin, and P. A. Adam. J Lab Clin Med89: 285-294, 1977). Therefore, the explanation for the discrepancy in calculated CHO(EXO) remains to be fully understood.     

The effect of methyl palmoxirate on incorporation of [U-14C]palmitate into rat brain

We examined the dose response, time course and reversibility of the effect of methyl 2-tetradecylglycidate (McN-3716, methyl palmoxirate or MEP), an inhibitor of -oxidation of fatty acids, on incorporation of radiolabeled palmitic acid ([U-¹⁴C]PA) from plasma into brain lipids of awake rats. MEP (0.1, 1 and 10 mg/kg) or vehicle was administered intravenously from 10 min to 72 hr prior to infusion of [U-¹⁴C]PA. Two hr pretreatment with MEP (0.1 to 10 mg/kg) increased brain organic radioactivity 1.2 to 1.8 fold and decreased brain aqueous radioactivity by 1.2 to 3.0 fold when compared to control values. At 10 mg/kg, MEP significantly increased brain organic fraction from 40% in controls to 85%, 30 min to 6 hr pretreatment, and resulted in a redistribution of the radiolabeled fatty acid toward triacylglycerol. MEP changed the lipid/aqueous brain ratio of incorporated [U-¹⁴C]PA from 0.67 to 5.7. The incorporation rate coefficient, k^*, was significantly increased by MEP (10 mg/kg) at 2 hr (31%), 4 hr (59%) and 6 hr (34%). All effects were reversed by 72 hr, consistent with a half-life of 2 days for carnitine palmitoyl transferase I. These results indicate that intravenous MEP may be used with [1-¹¹C]palmitic acid for studying brain lipid metabolism in vivo by positron emission tomography, as it significantly reduces the large unincorporated aqueous fraction that would result in high background radioactivity.     

[14C]acetylcholine synthesis and [14C]carbon dioxide production from [U-14C]glucose by tissue prisms from human neocortex.

1. [14C]Acetylcholine synthesis and 14CO2 production from [U-14C]glucose has been measured in tissue prism preparations from human neocortex. 2. Electron micrographs of prisms from human and rat neocortex show that both contain intact synaptic endings with evenly-distributed vesicles and normal-appearing mitochondria, but only poorly preserved cell body structure. 3. Synthesis of [14C]acetylcholine in prisms from rat neocortex is similar to estimates for turnover in vivo. Synthesis in prisms from human neocortex is 18% of that in rat tissue and 64% of that in tissue from baboon neocortex for incubations performed in 31 mM-K+. 4. Investigations of prisms prepared from rat brains stored at 37 degrees C after death revealed that synthesis of [14C]acetylcholine in the presence of 31 mM-K+ was greatly decreased within 30 min of post-mortem incubation, whereas synthesis at 5 mM-K+ and production of 14CO2 at both K+ concentrations were only significantly affected after longer periods. Changes were similar in neocortex and striatum. Thus human autopsy material is unlikely to be suitable for use with this system. 5. Investigations using animal models suggest that [14C]acetylcholine synthesis and 14CO2 production are not affected by surgical or anaesthetic procedures. 6. Neither [14C]acetylcholine synthesis nor 14CO2 production in human prisms was significantly changed with age between 15 and 68 years. 7. Samples from patients with the dementing condition Alzheimer's disease showed a significant decrease in [14C]acetylcholine synthesis to 47% of normal samples and a significant increase of 39% in production of 14CO2.     

Enzymatic synthesis of beta-[U-14C]alanine and D-[1,2,3-14C]pantothenate of high specific radioactivity

β-[U-¹⁴C]Alanine can be synthesized in >95% yield from l-[U-¹⁴C]aspartic acid using the aspartate 1-decarboxylase of Escherichia coli and converted to d-[1,2,3-¹⁴C]pantothenate in a 10–20% yield using the pantothenate synthetase of E. coli. Sufficiently pure preparations of both enzymes are readily obtained.     

Intermediates of peroxisomal beta-oxidation of [U-14C]hexadecanedionoate. A study of the acyl-CoA esters which accumulate during peroxisomal beta-oxidation of [U-14C]hexadecanedionate and [U-14C]hexadecanedionoyl-mono-CoA.

1. The oxidation of [U-14C]hexadecanedionoyl-mono-CoA was stimulated by CoA, by carnitine in the absence of CoA and by the presence of an NAD(+)-regenerating system. 2. Substrate inhibition was observed with respect to [U-14C]hexadecanedionoyl-mono-CoA at concentrations greater than 35 microM. 3. Acetyl-CoA and the dicarboxyl-CoA esters of chain length C6-16 were detected by HPLC under standard incubation conditions. 4. In the absence of the NAD(+)-regenerating system, 2-enoyl-CoA and 3-hydroxacyl-CoA esters were detected. 5. In general, the peroxisomal beta-oxidation of dicarboxylates is very similar to that of monocarboxylates [Bartlett, K., Hovik, R., Eaton, S., Watmough, N. J. & Osmundsen, H. (1990) Biochem. J. 270, 175-180] except that chain shortening does not proceed beyond C6. 6. We conclude that the peroxisomal beta-oxidation of dicarboxylates is regulated by the redox state of the peroxisomal matrix and CoA availability.     

Effect of evisceration on the disposal of [14C] palmitate in the rat.

The utilization in vivo of [1-(14)C] palmitate was studied in hepatectomized-nephrectomized rats and their sham-operated controls. After i.v. injection of the tracer, the [14C] lipids in plasma disappeared more slowly in eviscerated animals than in their controls. More label reappeared in plasma as esterified fatty acids in the latter group. At 30 min after the tracer, the amount of label found in the lipidic fraction of carcass and heart was much greater in eviscerated animals than in their controls although the percentile distribution of labelled lipidic fractions remained stable, a considerable proportion being present in the esterified fatty acid form. On the basis of these findings, the rapid increase in the plasma levels of FFA in eviscerated animals must be the result of augmented lipolytic activity more than reduced utilization of these metabolites.     

Formation of ketone bodies from [14C]palmitate and [14C]glycerol by tissues from ketotic sheep

Labelled ketone bodies were produced readily from [U-(14)C]palmitate, [2-(14)C]palmitate and [1-(14)C]glycerol by sheep rumen-epithelial and liver tissues in vitro. On a tissue-nitrogen basis, both tissues had similar capacities for ketogenesis. Palmitate was a ketogenic substrate in both rumen-epithelial tissue and liver, and more of its (14)C appeared in ketone bodies than in the (14)CO(2) liberated. Glycerol was actively metabolized to ketone bodies, but more readily underwent complete oxidation to carbon dioxide; this complete oxidation was most pronounced in rumen-epithelial tissue from ketotic ewes. These experiments with labelled compounds confirm earlier observations that rumen-epithelial tissue, like liver, actively forms ketone bodies from long-chain fatty acids and show further that normal rumen-epithelial tissue can convert palmitate into ketone bodies as readily as into carbon dioxide. Free glycerol, which is metabolized only by liver tissue in non-ruminants, is also metabolized by rumen epithelium. The rumen epithelium thus has unique metabolic capacity among extrahepatic tissues.     

Enzymatic production of the lignin precursor trans-[U-14C]cinnamic acid from l-[U-14C]phenylalanine using l-phenylalanine ammonia-lyase

An enzymatic method using phenylalanine ammonia-lyase (l-phenylalanine ammonia-lyase, EC 4.3.1.5) for the rapid conversion of l-[U-¹⁴C]phenylalanine to the deaminated lignin precursor trans-[U-¹⁴C]cinnamic acid is described. The method produces an experimentally useful ¹⁴C-labelled deaminated lignin precursor unavailable from radiochemical supply companies.     

The time course of glucagon action on the utilization of [U-14C]palmitate by isolated hepatocytes

The time course of glucagon action on the utilization of [U-¹⁴C]palmitate by isolated hepatocytes was studied. Ten minutes incubation of the cells after hormone addition was required in order to observe increased oxidation and decreased esterification of the labeled palmitate. The acid-soluble, labeled oxidation products could be separated into two main fractions, glucose and ketone bodies. Initially, glucagon directed the flux of radioactivity toward glucose and CO₂. After prolonged incubation in the presence of glucagon, labeled ketone bodies, as well as labeled glucose and ¹⁴CO₂, were increased. This effect was most marked as regards glucose. The results indicate that glucagon induces a rapidly onset stimulation of the rates of Krebs cycle and gluconeogenesis, while increased oxidation and decreased esterification of palmitate are time-delayed corresponding to the establishment of a lower level of glycerophosphate. About 10% of the glucose carbon formed by gluconeogenesis originated from the fatty acid when cells from fasted rats were incubated in the presence of alanine and [U-¹⁴C]palmitate.     

Ethanol potentiates the inhibitory effect of D-galactosamine on protein synthesis in isolated hepatocytes

The effect of the combined addition of D-galactosamine and ethanol on hepatic protein synthesis was studied in isolated mouse hepatocytes. 2.5 mM D-galactosamine or 40 mM ethanol alone caused slight or no inhibition of amino acid incorporation into proteins. However, a profound inhibition (about 80%) was observed if D-galactosamine of the same dose was added after a preincubation of the cells with 40 mM ethanol and vice versa. It shows that there is a strong mutual potentiation between D-galactosamine and ethanol in the inhibition of protein synthesis.