Synthesis of UDP-N-[1-14C]acetyl D-glucosamine and UDP-N-[1-14C]acetyl-D-galactosamine from [1-14C]acetate

Procedures for the preparation of UDP-N-[1-14C]acetyl-D-glucosamine and UDP-N-[1-14C]acetyl-D-galactosamine with very high specific activities are described. The overall yield based on the amount of [1-14C]acetate used is greater than 80%. The N-acetyl-D-glucosamine-alpha-1-phosphate used in this synthesis is prepared by phosphorylation of tetraacetyl-D-N-acetylglucosamine with crystalline phosphoric acid. N-acetyl-D-glucosamine-alpha-1-phosphate is then deacetylated in anhydrous hydrazine with hydrazine sulfate as a catalyst. D-glucosamine-alpha-1-phosphate is N-acetylated with [14C]acetate using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as the coupling agent. The acetylated product is coverted to the UDP derivative with yeast UDP-N-acetyl-D-glucosamine pyrophosphorylase. UDP-N-[1-14C]acetylgalactosamine is prepared by acetylation of UDP-galactosamine using [1-14C]acetate and N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline. UDP-galactosamine is prepared enzymatically using galactokinase and galactose-1-phosphate uridyltransferase. The labeled products, isolated and characterized by ion-exchange and paper chromatography, were active as substrates in glycosyl transferase systems.     

Compartmentation of [14C]glutamate and [14C]glutamine oxidative metabolism in the rat hippocampus as determined by microdialysis

Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free-moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect 14CO2 generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min-fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced 14C-amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [14C]glutamate and [14C]glutamine were 59 +/- 18 and 2.1 +/- 0.5 dpm/pmol, respectively. The initial infused specific activities for [U-14C]glutamate and [U-14C]glutamine were 408 +/- 8 and 387 +/- 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [14C]glutamine in the interstitial space when [14C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.     

Isoniazid-induced inhibition in the biotransformation of [14C] diphenylhydantoin in rat

The effect of isoniazid (0,10,20 and 40 mg/kg; i.v.) on the biliary and urinary excretion of [14C] diphenylhydantoin (DPH, 50 mg/kg; i.v.) was investigated in biliary-fistulated rats during the first 5 hours. Isoniazid cause a dose-dependent reduction in the total biliary excretion of 14 C without affecting its output in urine. After 5 h, maximal levels of radioactivity were recorded in the liver, followed by heart and brain. The glucuronides of DPH metabolites accounted for most of the 14C in 5 h pooled bile, less than 1% of the injected dose was excreted as unmetabolized DPH. Isoniazid administration caused the following significant dose-related changes in biliary excretion: (a) increased excretion of the unconjugated principal DPH metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantion (HPPH); (b) decreased excretion of HPPH-glucuronide; and (c) increased excretion of the glucuronide conjugates of the polar metabolites of DPH. The results suggest that the isoniazid-induced elevation of DPH-derived 14C in blood and its marked accumulation in brain, heart and liver is due to inhibition of p-hydroxylation of DPH and the glucuronidation of HPPH.     

Measuring DNA synthesis rates with [1-13C]glycine

We have devised and evaluated a stable-isotopic method for measuring DNA synthesis rates. The probe is [1-13C]-glycine that is incorporated into purines via de novo biosynthesis. The human hepatoma cell line HEP G2 was grown in medium containing [1-13C]glycine, the cells were harvested at various times, and the DNA was extracted. Following hydrolysis to the nucleosides, a reversed-phase HPLC separation was used to provide separate peaks for deoxythymidine (dT), deoxyadenosine (dA), and deoxyguanosine (dG). The HPLC effluent was continuously fed into a chemical reaction interface and an isotope ratio mass spectrometer (HPLC/CRI/IRMS). The isotope ratio of the CO2 produced in the CRI was used to monitor for enrichment. The cells were grown continuously for 5 days in labeled medium and also in a 1-day pulse labeling experiment where the washout of label was observed for the subsequent 9 days. As predicted from the role of glycine in de novo purine biosynthesis, the isotope ratio of the pyrimidine dT did not change. However, for the two purines, dA and dG, the characteristic log growth behavior of the cells was observed in their 13C/12C ratios and good agreement in the doubling time was obtained for each type of experiment. Parallel experiments that measured the HEP G2 doubling time in culture using tritiated thymidine incorporation and direct cell counts were carried out compare to our new method with established ones. We believe that the use of [1-13C]-glycine and the HPLC/CRI/IRMS is a highly sensitive and selective approach that forms the basis of a method that can measure DNA synthesis rates using a nonradioactive, nontoxic tracer.     

Synthesis and excretion profile of 1,4-[14C]phenylenebis(methylene)selenocyanate in the rat

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) inhibits chemically induced tumors in several laboratory animal models. To understand its mode of action, we synthesized p-[14C]XSC, examined its excretion pattern in female CD rats and also the nature of its metabolites. p-[14C]XSC was synthesized from alpha,alpha-dibromo-p-[ring-14C]xylene in 80% yield. The excretion profile of p-[14C]XSC (15.8 mg/kg body wt, 200 microCi/rat, oral administration, in 1 ml corn oil) in vivo was monitored by measuring radioactivity and selenium content. On the basis of radioactivity, approximately 20% of the dose was excreted in the urine and 68% in the feces over 3 days. The cumulative percentages of the dose excreted over 7 days were 24% in urine and 75% in feces, similar to excretion rates of selenium. According to selenium measurement, <1% of the dose was detected in exhaled air; radioactivity was not detected. Only 15% of the dose was extractable from the feces with EtOAc and was identified as tetraselenocyclophane (TSC). Most of the radioactivity remained tightly bound to the feces. Approximately 10% of this bound material converted to TSC on reduction with NaBH4. Organic soluble metabolites in urine did not exceed 2% of the dose; sulfate (9 % of urinary metabolites) and glucuronic acid (19.5% of urinary metabolites) conjugates were observed but their structural identification is still underway. Co-chromatography with a synthetic standard led to the detection of terephthalic acid (1,4-benzenedicarboxylic acid) as a minor metabolite. The major urinary conjugates contained selenium. Despite the low levels of selenium in the exhaled air, the reductive metabolism of p-XSC to H2Se cannot be ruled out. Identification of TSC in vivo indicates that a selenol may be a key intermediate responsible for the chemopreventive action of p-XSC.     

Metabolism of [1-(14)C] and [2-(14)C] leucine in cultured skin fibroblasts from patients with isovaleric acidemia. Characterization of metabolic defects

Leucine metabolism in cultured skin fibroblasts from patients with isovaleric acidemia was compared with that in normal fibroblasts and in cells from patients with maple syrup urine disease using [1-(14)C] and [2-(14)C] leucine as substrates. Inhibitory effects of methylenecyclopropylacetic acid on leucine metabolism in normal cells were also investigated. Production of 14CO2 from [2-(14)C] leucine was very reduced (96-99%) in both types of mutant cells. Radioactive isovaleric acid accumulated in assay media with isovaleric acidemia cells but not in those with maple syrup urine disease cells. Unexpectedly, 14CO2 production from [1-(14)C] leucine was partially depressed (80%) in isovaleric acidemia cells whereas in maple syrup urine disease cells it was strongly depressed (99%) as expected. These two mutant cells were clearly distinguished by detection of 14C-isovaleric acid accumulation after incubation with [2-(14)C] leucine. A pattern of inhibition of leucine oxidation similar to that seen in isovaleric acidemia cells was induced in normal cells by the addition of 0.7 mM methylenecyclopropylacetic acid to the assay medium. The partial inhibition of [1-(14)C] leucine oxidation seen in isovaleric acidemia cells and also in normal cells in the presence of the inhibitor appears to be, at least in part, due to an accumulation of isovalerate in the cells. Isovaleric acid (5-10) mM) inhibited [1-(14)C] leucine oxidation 32-68% when added to the assay medium with normal cells. Addition of flavin adenine dinucleoside to culture medium or assay medium or both did not restore oxidation of either leucine substrate in isovaleric acidemia cells.     

The incorporation of [14C]glucosamine into dolichol diphosphate N-acetyl[14C] glucosamine by unbroken liver cells in culture

Urinary testosterone and epitestosterone were assayed in 60 men: 7 normals and 53 patients with chronic prostatitis (of these 8 patients had prostatis free of complications, 45 had prostatitis with disturbances of generative and copulative functions). In 73.1% of patients considerable reduction of testosterone excretion was revealed. Reduction of testicular endocrine function is in direct correlative dependence on severity of clinical symptoms, duration of disease and form of chronic prostatis. Disturbances of genital hormone metabolism are of considerable importance in case of chronic prostatitis and its complications.     

Comparison of the one-gram d-[14C]xylose breath test to the [14C]bile acid breath test in patients with small-intestine bacterial overgrowth

In twelve patients with culture-proven bacterial overgrowth of the small intestine, the ability of a newly-developed one-gram d-[14C]xylose breath test to detect bacterial overgrowth was compared to that of the [14C]bile acid breath test. All patients manifested excessive production of breath 14CO2 after the administration of one gram [14C]xylose, with 83% of the patients being abnormal within the first hour of testing. In contrast, during the [14C]bile acid breath test, four of the twelve patients had no period of excessive 14CO2 production (above the 95% confidence range of controls). Nutrient malabsorption (fat, cobalamin, xylose) was seen with both true-positive and false-negative bile acid breath tests. The one gram [14C]xylose breath test, utilizing a substrate with more predominant absorption in the proximal small intestine and which can be catabolized by Gram-negative aerobic bacteria, appears to have a greater degree of sensitivity and specificity than the bile acid breath test in detecting the presence of small-intestine bacterial overgrowth.     

Determination of 14CO2 in breath and 14C in stool after oral administration of cholyl-1-[14C]glycine: clinical application

Twenty patients with intestinal bacterial overgrowth and 20 control subjects were investigated for bile acid deconjugation, by measuring 14CO2 in the breath after cholyl-1-[14C]glycine administration. 14CO2 output/24h was 11.0 +/- 5.2% (mean +/- SD) in controls and 54.2 +/- 14.0% (mean +/- SD) in bacterial-overgrowth patients (P less than .001). 14CO2 excretion rate in 12h, when normalized to 100% of the dose at the 12th hour, gave an even finer discrimination between the two groups (no false responses). 14C in stool, analyzed in 20 malabsorption patients and 20 controls by two different techniques, was 6.6 +/- 4% and 31.38 +/- 21.7% (mean +/- SD), respectively. Results by the two different techniques described here correlated well (r = .99). Bile acid malabsorption was in reasonable agreement (r = .67) with percentage of "chenoid" (chenodeoxycholic acid plus ursodeoxycholic acid) in the stool by gas-liquid chromatography; a poorer correlation was observed when "chenoid" plus "choloid" (cholic acid plus its epimers) were plotted vs. -4C in stool (r = .57, n = 15).     

Disposition of [14C]avitriptan in rats and humans

Avitriptan is a new 5-HT1-like agonist with abortive antimigraine properties. The study was conducted to characterize the pharmacokinetics, absolute bioavailability, and disposition of avitriptan after intravenous (iv) and oral administrations of [14C]avitriptan in rats and oral administration of [14C]avitriptan in humans. The doses used were 20 mg/kg iv and oral in the rat, 10 mg iv in humans, and 50 mg oral in humans. The drug was rapidly absorbed after oral administration, with peak plasma concentrations occurring at 0.5 hr postdose. Absolute bioavailability was 19.3% in rats and 17.2% in humans. Renal excretion was a minor route of elimination in both species, with the majority of the dose being excreted in the feces. After a single oral dose, urinary excretion accounted for 10% of the administered dose in rats and 18% of the administered dose in humans, with the remainder excreted in the feces. Extensive biliary excretion was observed in rats. Avitriptan was extensively metabolized after oral administration, with the unchanged drug accounting for 32% and 22% of the total radioactivity in plasma in rats and humans, respectively. Plasma terminal elimination half-life was approximately 1 hr in rats and approximately 5 hr in humans. The drug was extensively distributed in rat tissues, with a tendency to accumulate in the pigmented tissues of the eye.     

Biodegradability of [14C]methylcellulose by activated sludge

Three Methocel methylcellulose ethers of 1.9 degree of substitution with [14C]methyl labels were shown to be biodegradable using batch-type activated sludge tests. The maximum rate for conversion to 14CO2, attained after 1 week, was only 0.62 mg of methylcellulose/g of mixed liquor volatile solids per day. In 20 days, 55 to 73% of the radioactivity had been removed from solution as 14CO2, and the suspended solids contained 12 to 15% of the original radioactivity. Only 4% of the original methylcellulose appeared to be polymeric after the 20-day period. Thin-layer chromatography of supernatant liquid indicated at least two degradation products.     

Interleukin-1 beta inhibits the intestinal transport of [14C] 3-O-methylglucose in the rat

Interleukin-1 (IL-1) is known to be a hypoglycemic cytokine, but its mechanism of action is still unknown. Since the blood glucose levels depend on the amount of glucose entering and leaving the circulation, this work was conducted to test the hypothesis that the hypoglycemia observed with IL-1beta might result, at least partially, from a reduced intestinal glucose absorption. Male Sprague Dawley rats were injected intraperitoneally (i.p.) with IL-1beta, and a jejunal segment was perfused with [14C] 3-O-methylglucose for 5, 15, 25 and 40 min. Our results showed that IL-1beta significantly inhibited the mucosal uptake of this hexose and reduced its intestinal retention. The time course and the dose response effect for this cytokine were also determined. Studies on the effect of IL-1beta on the activity of the intestinal Na+-K+ ATPase demonstrated a significant inhibition of the pump. The effect of IL-1beta on the hexose transport across the brush border membrane may thus be attributed to its inhibitory effect on the Na+-K+ ATPase.     

Metabolism of [14C]paracetamol and its interactions with aspitin in hamsters

1. The metabolism of [14C]paracetamol (150 mg/kg) and its interactions with aspirin (200 mg/kg) were studied in male hamsters. 2. Aspirin was found to slow the rate of paracetamol absorption from the gastro-intestinal tract, but did not affect the rate of elimination. 3. Metabolism studies showed that greater than 80% of the radioactivity was excreted in the urine in 24 h. Paper chromatography of the urine separated the radioactivity into five peaks, four of which were identified as paracetamol and its glucuronide, sulphate and mercapturate conjugates. 4. The other peak, comprising of less than 10% of the total radioactivity, was a mixture of two or more other metabolites. A major component was isolated and characterized as methyl 2-hydroxy-5-acetamidophenyl sulphone. 5. Aspirin inhibited the metabolism of paracetamol by the sulphate conjugation pathway.     

Pharmacokinetics, bioavailability and tissue residues of [14C]isometamidium in non-infected and Trypanosoma congolense-infected Boran cattle

In this paper, pharmacokinetics, bioavailability and tissue residues are reported in non-infected and Trypanosoma congolense-infected Boran steers following either intravenous or intramuscular injection of [14C]isometamidium at a dose rate of 1 mg kg-1 body weight. Two differently labelled compounds of isometamidium were used; 6-14C (ISMM-1) and ring-U-14C (ISMM-2). The cattle were divided into 5 groups: group 1 consisted of 3 non-infected cattle treated with ISMM-1 by intravenous injection; group 2 consisted of 2 non-infected cattle treated with ISMM-1 by intramuscular injection; group 3 consisted of 2 Trypanosoma congolense-infected cattle given similar treatment as group 2 cattle; group 4 consisted of 3 non-infected and group 5 of 2 infected cattle treated with ISMM-2 by intramuscular injection. Radioactivity was measured in plasma, urine, faeces and tissues, and drug concentrations were calculated. Data obtained following i.v. treatment were best described by tri-exponential equations with half-lives of 0.13, 1.22 and 120.7 h. Bioavailability of the intramuscular dose was 58% in group 2 cattle. The major route of excretion was in faeces. Approximately 80% of the intravenous dose given was excreted within 21 days out of which only 18% was through urine. Total residues accounted for approximately 15% the total dose given. Drug residues remained high in organs with excretory functions including the liver and kidneys.