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.     

Effect of aeromonas salmonicida (furunculosis) infection on the incorporation of [1-14C]acetate into lipids of trout liver

[1-(14)C]acetate is incorporated into lipids more rapidly by liver from Aeromonas salmonicida-infected trout than by normal trout liver, both in vitro and in vivo. The increase was, in general, about twofold to less than threefold.     

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.     

Retarded incorporation of [14C]mannose into thyroglobulin of human thyrotoxic thyroid glands

In vitro studies using thyroid slices from human non-toxic goitres and from thyrotoxic glands show retarded incorporation of [14C]mannose into the 19S protein of thyrotoxic glands. This was not found using [14C]galactose with thyrotoxic glands or using either labelled sugar with slices from non-toxic goitres. Experiments with thyroid tissue from rats on a variety of treatment regimes such as iodine supplements, carbimazole alone or with iodine supplements did not show this differential delay of [14C]mannose incorporation. This suggests that there may be some abnormality of carbohydrate incorporation into thyroglobulin in thyrotoxicosis.     

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.     

14C]leucine incorporation into proteins of pancreatic juice in rats fed soya-bean flour

1. The effect of a diet containing a trypsin inhibitor on the incorporation of radioactively labelled leucine into the pancreatic proteins secreted during stimulation with cholecystokinin-pancreozymin (CCK) was studied in rats. 2. The total output of protein was significantly greater in the rats given raw soya-bean flour (RSF) compared with those given heat-inactivated soya-bean flour (HSF) (controls) in response to the sub- and supramaximal stimulation with CCK, but similar responses were obtained to maximal stimulation with CCK. Total protein output decreased continuously with time after reaching peak values at 90--120 min after the start of stimulation with CCK. 3. The total output of radioactively labelled protein in RSF-fed rats was not different from that of the controls with sub- and supramaximal dose rats of CCK, but was significantly lower than that of the controls in response to the dose rate of CCK which produced maximal rates of pancreatic secretion. 4. The specific activity of radioactively labelled protein increased continuously, while the output attained a constant rate during stimulation with all doses of CCK. 5. We concluded that feeding the trypsin inhibitor-containing diet led to increased secretion of stored pancreatic protein, while secretion of newly synthesized protein was not altered. During the course of prolonged stimulation with CCK, irrespective of diet, there was increasing secretion of the newly synthesized protein compared with the pre-existing stored proteins of the pancreas, it was unable to compensate for the decreased secretion of pre-formed protein.     

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.     

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).     

20-[18F]fluoroarachidonic acid: tissue biodistribution and incorporation into phospholipids

The in vivo behavior of 20-[18F]fluoroarachidonic acid (18F-FAA) was investigated to evaluate its potential use as a radiotracer for studying the regional brain and heart lipid metabolism by positron emission tomography (PET). Tissue biodistribution studies in rats have revealed that 18F-FAA has a high uptake in the liver and lung, thus probably reflecting the metabolism, and is accompanied by both low in vivo defluorination and low blood levels. At 30 min postinjection, the uptake in the brain and heart reached values of 0.26 +/- 0.02 and 1.22 +/- 0.58% dose/g, respectively, with ratios to the blood radioactivity of 1.04 and 4.88, respectively. Lipid extraction at 30 min postinjection showed that 39% of the brain radioactivity was in the organic phase whereas the organic phase from heart tissue contained 73% of the total radioactivity. A TLC analysis demonstrated that 18F-FAA was mainly bound to phospholipids in the brain and heart tissue as expected. Based on the findings of this study, the utility of 18F-FAA as an in vivo tracer for cerebral phospholipid studies appears to be limited because of its relatively high radioactivity in the aqueous brain fraction. However, our findings do suggest that this agent might be useful as a tool for studies of cardiac phospholipid turnover, even though it demonstrated a poor heart-to-lung and heart-to-liver contrast.     

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.     

Temporal dynamics and regional distribution of [14C]serine uptake into mouse brain

In order to examine the uptake of L-serine into brain structures and brain metabolic compartments, L-[U-14C]serine was injected into tail vein of mice. The uptake was examined 30 min, 90 min, 3 h and 5 h after injection by both quantitative autoradiography of coronal brain sections and by biochemical analysis. Brain radioactivity was extracted and partitioned into protein associated pellets, metabolites soluble in aqueous phase and lipids soluble in the organic phase. Most of the radioactivity was found in the aqueous phase, about 10% was incorporated into lipids. Among phospholipids the highest label was found in phosphatidylserine, then in phosphatidylethanolamine and in phosphatidylcholine, it amounted to 52%, 30% and 18% of label by 90 min after injection, respectively. The brain distribution of L-serine uptake resembled that described for strychnine-insensitive [3H]glycine binding, with cortical structures being preferentially labelled.     

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.     

Incorporation of (1-14C)oleic acid and (1-14C)arachidonic acid into lipids in the subcellular fractions of mouse brain

The response of the astroglial population of the dentate gyrus molecular layer to removal of that region's primary afferent was investigated using Cajal's gold sublimate method. Deafferentation caused the astrocytes to hypertrophy, an effect which was detectable at 24 hr and maximal at 72-96 hr post-lesion. Following this, the astroglia entered a lengthy period of gradual atrophy. Counts of the astrocytes in the various sublayers of the molecular layer led to the conclusion that these cells migrate into denervated dendritic areas from neighboring, nondeafferented zones.