14C-labeled Acids Research Articles

Semi-reversible cross-linking. Synthesis and application of a novel heterobifunctional reagent.

The synthesis of a novel heterobifunctional cross-linking reagent, 3-(4-azido-2-nitrobenzoylseleno)propionic acid is described. The selenol ester function serves as a selective and reversible thiol reagent. Cross-linking is carried out photochemically via nitrene generation. The reagent is well suited for the identification of the environment of a defined cysteine residue according to the principle of semi-reversible cross-linking (Trommer, W.E., Friebel, K., Kiltz, H.-H. & Kolkenbrock. H. (1977) Adv. Exp. Med. Biol. 86A, 187-195). Cleavage of the thiol ester bond (the first anchor point) can be achieved by a variety of labels carrying an amino function thus giving rise to labelled amino acids or proteins in a distance of about 0.7 nm from the known cysteine residue. Optimal conditions were determined by reaction of 3-(4-azido-2-nitrobenzoylseleno)propionic acid and of its unsubstituted analog with glutathione and N alpha-benzoylglycyllysine. Nucleosidetriphosphate-adenylate kinase from pig muscle was chosen as an example to demonstrate the usefulness of the reagent. 14C-labelled aminobutyric acid was applied to cleave the thiol ester bond between the reagent and cysteine-25 leading to labelling of the N-terminal arginine peptide.

In vitro binding of 14C-labeled acidic compounds to serum albumin and their tissue distribution in the rat.

The acidic compounds, such as phenoxyacetic acids, substituted benzoic acids, or acetylsalicylic acid, were found to bind to bovine serum albumin (BSA). Among phenoxyacetic acids, the binding affinity to BSA was highest for 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), which was approximately 4-, 24-, and 160-fold greater than those for 2,4-dichlorophenoxyacetic acid (2,4-D), o-chlorophenoxyacetic acid (CPA), and phenoxyacetic acid (PAA), respectively. There were two bound to serum albumins of other mammalian species. The binding affinity varied among species and also depended on the chemicals. However, the order of binding affinity in the albumin of each species remained the same as observed in BSA with few exceptions. Blood/tissue ratios of 14C from rats dosed with these 14C-labeled acids were highly correlated with the logarithm of the binding affinity constantsaffinity constants.

Hydroxylation of cholic, chenodeoxycholic, and deoxycholic acids in patients with intrahepatic cholestasis

The metabolism of 14C-labeled chenodeoxycholic, cholic, and deoxycholic acids was studied in patients with intrahepatic cholestasis. Radioactively labeled metabolites were isolated from urine and were identified by gas-liquid chromatography-mass spectrometry. About 5% of the radioactivity was recovered in urine after administration of labeled chenodeoxycholic acid to a patient with mild intrahepatic cholestasis. In urine collected 0-24 hr after the injection, 20% of the radioactivity appeared in the combined glycine and taurine conjugate fractions, and the predominant metabolite in these fractions was identified as hyocholic acid. Eighty percent of the activity was eluted in the sulfate fraction presumably representing mainly sulfated chenodeoxycholic acid conjugates. Twenty percent of the radioactivity was recovered in urine following administration of labeled cholic acid to a patient with biliary cirrhosis and severe cholestasis. In urine collected on the fifth day, half of this radioactivity appeared in the glycine and taurine conjugate fractions, and 10% of this activity was present as tetrahydroxycholanoates. The major metabolites in this fraction were 3 alpha, 6 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta- and 1 xi, 3 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholanoic acids. The former compound constituted about 50% of the tetrahydroxycholanoates. Three additional minor tetrahydroxy bile acids were present, one of which was tentatively identified as 6 beta-hydroxycholic acid. About 5% of the radioactivity appeared in urine after oral administration of labeled deoxycholic acid to a patient with mild intrahepatic cholestasis. Twenty-two percent of the activity appeared in the glycine and taurine conjugate fractions isolated from urine collected on the second day after the administration. About 75% of this activity was associated with trihydroxycholanoates. The main metabolite was 1 beta-hydroxydeoxycholic acid with small amounts of, tentatively, 6 alpha-hydroxydeoxycholic acid.

Cholesterol and bile acid metabolism in obesity.

1. The present study was undertaken to determine the influence of obesity on bile acid kinetics and cholesterol balance in man. 2. Fourteen obese and normolipidaemic patients (160 +/- 6% of ideal body weight, mean +/- SEM) were studied under standardized dietary conditions. Bile acid kinetics, were determined with the aid of 14C-labelled cholic acid and chenodeoxycholic acid. Cholesterol balance was calculated as the sum of bile acid synthesis plus daily faecal excretion of neutral C27 steroids minus dietary intake of cholesterol. The results obtained were compared with previously published data on control subjects (n = 13). 3. The cholesterol balance was higher in the obese patients (2.61 +/- 0.27 mmol/day) than in the control subjects (1.78 +/- 0.22 mmol/day), owing to a higher excretion of neutral steroids. When expressed per kg of body weight the cholesterol balance was quite normal in the obese patients.

The synthesis of bile acid glucuronides by agarose-bound UDP-glucuronyltransferase

Lithocholic acid, chenodeoxycholic acid and deoxycholic acid but not cholic acid or the glycine or taurine conjugates of these bile acids are glucuronidated by agarose-bound UDPglucuronyltransferase from rat liver in the presence of UDPglucuronic acid. The identification of bile acid glucuronides is based on the specific enzymatic hydrolysis, incorporation of 14C-labeled glucuronic acid into the molecule and by mass spectrometry. Mass spectrometry indicates that bile acid glucuronides formed by agarose-bound UDPglucuronyltransferase are 3-glucuronides. The glucuronidation of lithocholic acid, chenodeoxycholic acid and deoxycholic acid is linear up to 45 min and at a protein concentration between 0.5 and 2.0 mg per assay. The pH optimum of the enzyme reaction is 7.0. The K m for lithocholic acid, chenodeoxycholic acid and deoxycholic acid, which are 0.040, 0.031 and 0.037 mM, respectively, are similar to those found in microsomal preparations. The use of agarose-bound UDP-glucuronyltransferase offers the possibility to synthesize bile acid glucuronides which are needed for kinetic studies in man and animal.

Gas-chromatographic determination of urinary oxalate.

We describe a simple, rapid method for determining urinary oxalate: isolation by precipitation with calcium chloride and conversion to dimethyl oxalate, which then is measured by gas chromatography. To each sample, tracer amounts of 14C-labeled oxalic acid are added, to determine the analytical recovery of urinary oxalate. Analytical recovery of [14C]oxalic acid added to urine specimens ranged from 15 to 95% (mean, 80%), and corrected recovery, based on calculation isotope-dilution techniques, ranged from 98 to 100%. The urinary excretion of oxalic acid by 18 normal men, ages 23 to 43 years, ranged between 9 and 23 mg/24h, with a mean value of 16 mg; that by 68 patients with small-bowel bypass ranged from 60 to 210 mg/24 h, with a mean of 127 mg.

Adenosine inhibition of gamma-aminobutyric acid release from slices of rat cerebral cortex.

1 The effect of purine compounds on the potassium-evoked release of 14C-labelled gamma-aminobutyric acid (GABA) has been studied in 400 micrometers slices of rat cerebral cortex in vitro. 2 Adenosine and adenosine 5' monophosphate (AMP) inhibited the release of GABA at 10(-5) to 10(-3) M. Adenosine triphosphate (ATP) produced a significant inhibition of release only at 10(-3) M. 3 Theophylline 10(-4) or 10(-3) M reduced the inhibitory effect of adenosine, but did not change basal release of GABA. 4 Dipyridamole 10(-5) M itself reduced evoked GABA release, but did not prevent the inhibitory effect of adenosine, implying that adenosine was acting at an extracellularly directed receptor. 5 Calcium removal or antagonism by verapamil reduced the evoked release of GABA, but adenosine did not produce any further reduction of the calcium-independent release. This may indicate that the inhibitory effect of adenosine on GABA release results from interference with calcium influx or availability within the terminals.

Characteristics of D- and L-alanine transport into mouse Ehrlich ascites tumor cells.

The present investigation was undertaken to examine the temperature dependence of the kinetic parameters for the uptake and exit of D- and L-alanine in Ehrlich ascites tumor cells, as one of a series of studies to account for the high in vivo uptake of radio-activity of 14C-labeled D-amino acids in various tumor cells. The Vmax values for D- and L-alanine uptake decreased with a fall in temperature. The activation energies for both isomers obtained from plots of In Vmax against 1/T suggested that these isomers were transported through a mediated process. On the other hand, Km for D-alanine uptake was only slightly dependent on temperature, whereas Km for L-alanine decreased with a fall in temperature. These temperature dependences of Km are discussed in comparison with those of Km for D- and L-leucine uptakes. Furthermore, it was observed that the exit rate of D-alanine was considerably slower than that of the L-isomer. The activation energies of the isomers obtained from plots of ln ke against 1/T (ke : the first-order rate constant for exit) suggested that free diffusion is predominant in the exit process of D-alanine, in contrast to that of the L-form. This slow exit of D-alanine may account in part for the high in vivo uptake of radioactivity of D-alanine-14C into Ehrlich tumor cells.

Enterohepatic circulation rates of cholic acid and chenodeoxycholic acid in man.

The rate of enterohepatic cycling of cholic acid and chenodeoxycholic acid was determined in five male subjects. Pool sizes were measured by isotope dilution technique after intraduodenal administration of 14C-labelled cholic and chenodeoxycholic acid. The hourly hepatic secretion rate of bile acids was determined by an intestinal perfusion technique. From these data the cycling frequency was calculated. Chenodeoxycholic acid circulated on an average 1.34 (range, 1.13--1.57) times faster than cholic acid, probably because chenodeoxycholic acid to a larger extent than cholic acid is absorbed from the proximal small intestine and thus partly bypasses the hepaticoileal circuit. This difference in cycling rate may have methodological as well as physiological implications.

Characterization of endogenous folate and incorporation of labelled folates into the brain of the South African fruit bat

Folate in the brain of the South African fruit bat consists of 10-formyltetrahydropteroylglutamic acid and the tri-, tetra- and penta- forms of 5-methyltetrahydropteroylglutamic acid. Following parenteral injection, only 5-[ 3H]-methyltetrahydropteroylglutamic acid was taken up by the brain, but none of a dose of 14C-labelled tetrahydropteroylglutamic acid was detectable. Only trace amounts of the 5-methyltetrahydropteroylglutamic acid were converted into the formyl compounds and a small amount of methyltetrahydropteroyltriglutamic acid appeared after 96 h. There was no significant difference in vitamin B-12-deficient animals.

Haem Synthesis in Sideroblastic Anaemia

Measurement of delta-aminolaevulinic acid (ALA) synthetase has been performed in bone marrow of 24 normal subjects and 20 patients with sideroblastic anaemia (five congenital, two pyridoxine-responsive, 12 primary acquired and one secondary form due to isoniazid therapy). ALA-synthetase activity without added pyridoxal-phosphate in vitro was decreased in four of the congenital cases, and in all 12 primary acquired cases. In two of these 16 cases with low ALA-synthetase activity and in a case with sideroblastic change secondary to isoniazid therapy, the enzyme activity was corrected to normal by pyridoxal-phosphate in vitro. ALA-synthetase activity was normal in both cases of pyridoxine-responsive anaemia while they were receiving pyridoxine therapy but in one of these cases, studied at repeated intervals after withdrawal of pyridoxine therapy, the bone marrow ALA-synthetase activity fell to very low levels unless pyridoxal-phosphate was added in vitro. In this case the Km of the enzyme for pyridoxal-phosphate was substantially greater than that of a control sample. Haem synthetase activity was reduced in the bone marrow of two of six cases of primary acquired sideroblastic anaemia and in one congenital case tested but was normal in the pyridoxine-responsive patient studied on pyridoxine therapy. Nevertheless, this patient showed reduced incorporation of 14C-labelled delta-aminolaevulinic acid into bone marrow haem. It is concluded that both congenital and primary acquired sideroblastic anaemia are a heterogenous group of disorders, with different defects in haem synthesis, reduced activity of delta-aminolaevulinic acid synthetase being common but not invariable in both types. Pyridoxine-responsive (congenital) sideroblastic anaemia may be due to the presence of an abnormal ALA-synthetase apoenzyme which requires excessive amounts of the coenzyme, pyridoxal-phosphate, to achieve normal activity; other abnormalities of haem synthesis may also be present.

Ascorbic acid metabolism in geranium and grape

l-Ascorbic acid-[UL- 14C] has been used to follow the appearance of 14C-labeled oxalic acid and tartaric acid as metabolic products of oxidative cleavage of ascorbic acid in geranium apices ( Pelargonium crispum). The enantiomeric specificity of ascorbic acid metabolism was established in geranium by comparing the incorporation of d- and l-ascorbic acid-[6- 14C] in the presence of l-ascorbic acid-[4- 3H]. l-Ascorbic acid-[4- 3H] has been used to demonstrate the retention of 3H during biosynthesis of l-(+)-tartaric acid in the geranium and its exchange with water during biosynthesis of l-( +)-tartaric acid in the grape.

Developmental aspects of maleic acid-induced inhibition of sugar and amino acid transport in the rat renal tubule.

The transport of alpha-aminoisobutyric acid and alpha-methyl-d-glucoside by isolated tubules from Sprague-Dawley rats at different stages of development follows a separate age-dependent pattern for each substrate. The effects of 6 M maleic acid on transport processes differ for amino acids and sugars and become manifest at distinct points during development. Maximum inhibition by maleic acid occurs at a time subsequent to maturity of these transport systems. In an effort to explain these transport phenomena, the uptake and metabolism of 14C-labeled maleic acid by the newborn or adult renal tubule was studied, showing significant binding by the tubule membrane, penetration of the cell by diffusion, and no conversion to 14CO2. Maleic acid has no demonstrable effect on the membrane-associated enzymes which are thought to play a role in the transport of small molecules.

Studies of the metabolism of 3-phenoxybenzoic acid in plants

The metabolism of 14C-labeled 3-phenoxybenzoic acid, PBA, has been studied in cotton, vine, broad bean, soya bean, pea, lettuce, and tomato, using abscised leaves. PBA was converted into a range of polar products by esterification with glucose (in cotton and other species) and with glucosylarabinose and glucosylxylose (especially in vine leaves). Uptake of the glucose ester itself by cotton leaves also led to conversion into a more polar conjugate, probably the glucosylarabinose ester, which was not detectable following uptake of PBA. When PBA was applied to the surface of intact cotton leaves, it was slowly converted into products similar to those above.

The metabolism of ethyl and n-propyl- p-hydroxybenzoate (“parabens”) in male cats

Metabolic studies conducted on the ethyl and n-propyl esters of 14C-labelled p-hydroxybenzoic acid orally administered to cats at a dose level equivalent to 130 mg kg of the parent acid, have shown that from both of these compounds radioactivity is rapidly excreted in the urine within 72 h. Examination of the urine of these animals by thin-layer and DEAE-Sephadex A-25 chromatography revealed two major metabolites, one of which was identified as p-hydroxyhippuric acid, and the other as free p-hydroxybenzoic acid. A parallel study on the metabolism of a single oral dose of 14C-labelled p-hydroxy-enzoic acid of 130 mg kg showed that, as with the esters, substantially all of the radioactivity was excreted in the urine within 72 h. However, only one urinary metabolite, p-hydroxyhippuric acid was found in this experiment.

Transfer and dynamics of uric acid in the pregnant rhesus monkey: II. A mathematical model

The objective of the present study was to develop a mathematical model of the dynamics of uric acid between fetal and maternal compartments in the term pregnant rhesus monkey. In 3 different animals 14C-labeled uric acid was injected into the fetal circulation, the amniotic fluid and the maternal circulation, respectively. In one experiment no uric acid was administered and the fetus was deliberately killed at the beginning of the experiment. Samples of fetal and maternal blood, maternal urine and amniotic fluid were collected at regular intervals. Semilogarithmic time-activity curves were constructed and time constants were determined. An open four-compartment model (fetal-placental plasma, fetal-placental interstitial space, amniotic fluid and maternal plasma) was applied to describe the intercompartmental dynamics of uric acid. Transplacental clearance was approx. 1 ml · min −1 in both directions, maternal renal clearance was about 17 ml · min −1. These results and the calculated values of the other intercompartmental clearances support earlier results, obtained with the steady infusion method. Uric acid concentrations in amniotic fluid and fetal plasma appeared to increase significantly during the experiments. The rise in amniotic fluid levels can only be explained by accepting a yet undefined compartment in which uric acid is produced and cleared directly into the amniotic cavity. It is speculated that this additional compartment could be the fetal lung.

Relationship Between the Axenic Phenotype and Sensitivity to -Aminocarboxylic Acids in Dictyostelium discoideum

A previous suggestion that sensitivity to ω-aminocarboxylic acids in Dictyostelium discoideum is associated with the ability to grow axenically has been re-evaluated using new axenic strains and axenic derivatives of strain ax3 recombinant for linkage group II. It is now shown that the major gene for ω-aminocarboxylic acid sensitivity, designated oaaA., is not allelic to previously described axenic loci axeA and axeB, but that oaaA is closely linked to, or allelic with a third axenic gene axeC found in strains ax2 and ax3. The axeC phenotype is rapid growth in axenic medium. The oaaA locus is located on linkage group II. Genetic evidence suggests that the strains ax2 and ax3 now in existence share a common genetic background and may not represent independent isolates from the wild-type strain nc4. Studies with 14C-labelled ϵ-aminocaproic acid show that sensitivity to ω-aminocarboxylic acids is correlated with increased myxamoebal permeability to these compounds.

Transfer and dynamics of uric acid in the pregnant rhesus monkey: I. Transplacental and renal uric acid clearances

Data from the literature suggest that uric acid produced by the fetus could pass across the placenta and contribute to the observed increase in maternal plasma levels of uric acid in preeclamptic pregnancy. To investigate this hypothesis, fetal transplacental and renal uric acid clearances were estimated in 4 term pregnant rhesus monkeys by means of the steady infusion method using 14C-labeled uric acid. Allantoin clearances were determined in one pregnant monkey. Samples of maternal and fetal arterial blood were collected at regular intervals. The total amount of fetal urine produced during the experiment was collected at the end of the experiment. In addition, maternal endogenous renal uric acid and creatinine clearances were measured in 6 term pregnant monkeys. Fetal transplacental uric acid clearances appeared to be almost entirely limited by placental permeability and varied between 3.6 and 8.6 ml · min −1 · kg −1 of fetal weight; fetal renal clearances were between 0.11 and 0.20 ml · min −1 · kg −1. The allantoin clearances were found to be of the same magnitude. Maternal renal clearances of uric acid and creatinine were almost equal (mean 3.2 ± 0.6 and 3.0 ± 0.5 ml · min −1 · kg −1 of maternal weight, respectively). Extrapolation of these data to human preeclamptic pregnancy reveals that it is unlikely that fetal uric acid could significantly contribute to the maternal uric acid load.

Continuous measurement of 14C-labeled carbon dioxide and lactic acid by cultured cells grown in Leighton tubes

A procedure for continuous measurement of 14CO 2 production by cultured cells grown in Leighton tubes has been described. The apparatus developed also permits aliquots of the incubation medium to be taken during the experiments for analysis of labeled metabolites released into the solution. A simple method for determination of [ 14C]lactic acid in such aliquots has been described. The reproducibility and usefulness of the apparatus has been demonstrated by incubating fibroblasts with glucose labeled in the C-1 or C-6 position, and examining the effects of selected drugs on CO 2 and lactic acid production.

209 DEVELOPMENTAL ASPECTS OF MALEIC ACID INDUCED INHIBITION OF SUGAR AND AMINO ACID TRANSPORT IN THE RAT RENAL TUBULE

The transport of alpha-aminoisobutyric acid and alpha-methyl-D-glucoside by isolated renal tubules from Sprague-Dawley rats at different stages of development follows a separate age-dependent pattern for each substrate. The effects of 6 mM maleic acid on transport processes differ for amino acids and sugars and become manifest at distinct points during development. Maximum inhibition by maleic acid occurs at a time subsequent to complete maturation of these transport systems. In an effort to explain these transport phenomena, the uptake and metabolism of 14C-labelled maleic acid by the newborn and adult renal tubule was studied, showing significant binding by the tubule membrane, penetration of the cell by diffusion, and no conversion to ≥. Maleic acid has no demonstrable effect on the membrane-associated enzymes which are thought to play a role in the transport of small molecules. Though the mechanisms by which maleic acid inhibits the transport of sugars and amino acids in the adult rat tubule remains unexplained, it is clear that its effects are age-related and independent of the maturation of these transport systems. This observation is consistent with the delayed appearance of the Fanconi syndrome seen in human cystinosis and suggests that the progressive tubular dysfunction in this syndrome and in the maleic acid model is secondary to genetically directed intracellular metabolic changes expressed during development.