Radio-gas Chromatography Research Articles

George Alan Garton. 4 June 1922—13 May 2010

Alan Garton and his group at The Rowett Research Institute in Aberdeen made very significant and unique contributions to the understanding of differences in digestion, absorption and metabolism of dietary lipids (fats) and their importance to health in mammalian species, particularly farm animals, but also various aspects of lipid metabolism and health in humans. The group were the first to use commercially available gas chromatography and first, this side of the Atlantic, to employ thin-layer chromatography for lipid analyses. A special adaptation of radio-gas chromatography, developed in-house, also led to the definitive evidence that radio-labelled methylmalonyl-CoA, derived from propionic acid, particularly from grain-fed sheep, can be utilized for fatty acid synthesis. Alan always highlighted the relevance of the group's lipid research to human health and wellbeing, as seen by the role of branched-chain fatty acids in Refsum's disease and the later development of studies of the role of saturated and essential omega-3, -6 and -9 fatty acids, including conjugated linoleic acids and trans fatty acids, in human health and disease by the group.

11CH4 molecule production using a NaBH4 target for 11C ion acceleration

Solid-state materials suitable for use as proton irradiation targets were investigated for producing high-purity (11)CH4 molecules for heavy-ion cancer therapy. The radioactivity of gas produced by proton irradiation was measured for several target materials. Also, the radioactive molecular species of the produced gas were analyzed by radio gas chromatography. We found that 5 × 10(12) (11)C molecules could be collected by proton irradiation on a NaBH4 target. We also found that the (11)CH4 molecules were produced and collected directly from the irradiated target, owing to the hydrogen atoms bound in the solid-state NaBH4.

In vitro metabolism of (3H)-androstenedione in the rat epididymis and vas deferens.

The in vitro metabolism of (3H)-androstenedione in the epididymis and vas deferens of intact and castrated rats was investigated and the metabolites formed were identified by radio gas chromatography. Incubation of slices of caput epididymidis for 2 hr at 34 degrees C metabolised 90% androstenedione. Similar incubations of tissue samples from cauda epididymidis and vas deferens metabolized 60 and 25% of androstenedione respectively. The major metabolites formed in the epididymis were androstanedione (caput: 48%; cauda: 33%) and androsterone (caput: 35%; cauda: 13%). These metabolites appeared in much less concentration in the incubations with vas deferens (about 8% each). In general, conversion to testosterone and dihydrotesterone was low in all the three organs examined. Castration did not significantly alter the metabolic pattern in the caput epididymidis and vas deferens but promoted the formation of androsterone (38%) in the cauda epididymidis. The conversion of androstenedione, a weak androgen to testosterone, dihydrotestosterone and 3 alpha/3 beta-diols in the epididymidis and vas deferens of castrated rats may be of physiological significance. In addition, androsterone appears to be an important androgenic metabolite in the epididymis.

Silencing Geranylgeranyl Diphosphate Synthase inNicotiana attenuataDramatically Impairs Resistance to Tobacco Hornworm

In bioassays with artificial diets, the 17-hydroxygeranyllinalool diterpenoid glycosides (HGL-DTGs) of Nicotiana attenuata function as antifeedants for the plant's adapted herbivore, tobacco hornworm (Manduca sexta). To determine whether HGL-DTGs have a defensive function in planta, we suppressed HGL-DTG production by silencing the source of the geranylgeranyl diphosphates (GGPPs) required for geranyllinalool biosynthesis, a key intermediate. We used virus-induced gene silencing to suppress transcript levels of GGPP synthase gene (Naggpps) and farnesyl diphosphate (FPP) synthase gene (Nafpps), northern blotting and real-time polymerase chain reaction to quantify transcript accumulations, and radio gas chromatography to analyze prenyltransferase specificity. Silencing Nafpps had no effect on the accumulation of HGL-DTGs but decreased leaf steroid content, demonstrating that DTG-synthesizing enzymes do not use GGPP derived from FPP and confirming FPP's role as a steroid precursor. Unlike plants silenced in the phytoene desaturase gene (Napds), which rapidly bleached, Naggpps-silenced plants had reduced HGL-DTG but not carotenoids or chlorophyll contents, demonstrating that Naggpps supplies substrates for GGPP biosynthesis for HGL-DTGs, but not for phytoene or phytol. Expression of Naggpps in Escherichia coli revealed that the recombinant protein catalyzes the GGPP synthesis from isopentenyl diphosphate and dimethylallyl diphosphate. When fed on silenced plants, hornworm larvae gained up to 3 times more mass than those that fed on empty vector control plants or plants silenced in Nafpps, the trypsin protease inhibitor gene, or the putrescine N-methyltransferase gene. We conclude that HGL-DTGs or other minor undetected diterpenoids derived from GGPP function as direct defenses for N. attenuata and are more potent than nicotine or trypsin protease inhibitors against attack by hornworm larvae.

Radioisotopic tracing of methanol transformation using 11C-labelled methanol over copper ion-exchanged H-ZSM-5, H-Beta and H-MCM-41

Short half-life 11C-radioisotope tracer in methanol form was used to study the transformation of methanol over copper ion-exchanged H-ZSM-5, H-Beta and H-MCM-41 catalysts. The progresses of adsorption and desorption as well as methanol transformation were followed by radiodetection. The reaction products were analyzed by radio-gas chromatography. The GC analysis was completed with a radiodetector that could distinguish the radiolabelled derivates from other non-radioactive compounds, reagents or impurities. The radiodetector can be more sensitive to detect smaller amount of compound than the thermal conductivity detector or flame ionization detector since the intensity of a radio-signal is independent on thermal conductivity or ionization energy of compounds. The copper introduction into the catalysts had an inhibitory effect on the reaction to proceed further from the equilibration with the methanol and dimethyl ether resulting in hydrocarbons C3–C6. Presence of copper also allowed a possible reaction route for methanol to give formaldehyde and methyl formate.

A Novel Radioisotope Method for Studying Catalytic Transformations over Alumina, H-ZSM-5 and H-Beta Zeolite Catalysts: Investigation of Conversion of11C-Labeled Methanol to11C-Labeled Dimethyl Ether and Hydrocarbons

A novel radiochemical method for investigating the catalytic transformations of the 11C-radioisotope labeled methanol over H-ZSM-5 and H-Beta zeolite catalysts has been introduced. The catalysis process was monitored by gamma detectors and the 11C-labeled products were analyzed by radio-gas chromatography. The medium pore H-ZSM-5 and H-Beta zeolite catalysts were synthesized and characterized using X-ray powder diffraction, scanning electron microscope, nitrogen adsorption, X-ray fluorescency and FTIR spectroscopy. The investigations of 11C-labeled product distributions and reaction mechanism of the conversion of [11C]methanol over H-ZSM-5 and H-Beta zeolite catalysts have been elaborated in terms of structure and acidity of the catalysts. In microreactors the effect of natural carbon compounds from environment can be a disturbing effect for the detection of inactive carbon products. Applied radio detection method eliminates these disturbing effects and detects only 11C-labeled compounds during the whole catalytic process. In the study of the transformations of carbon compounds, besides the well known 14C tracer technique and 13C MAS NMR spectroscopy investigation, the 11C-method is a new, more sensitive and simple one to monitor the transformation of the starting 11C-labeled compound by radio detectors (gamma detector) and for analyzing the 11C-labeled products by radio-gas chromatography.

Investigations of cholesterol transformation during sewage treatment: relevance to estrogen formation pathways?

There is currently a great deal of concern over the observation of so-called estrogenic effects (specifically increases in the concentrations of the egg yolk precursor, vitellogenin) in male fish living in some UK rivers. The effects have been attributed to chemicals, including estrogenic steroids, which enter the rivers via sewage effluents. The origins of these estrogenic steroids in sewage may include contributions from the influents and possibly in situ transformation processes of other steroids occurring during sewage treatment. The present study examined the latter possibility. The bacterial metabolism of radiolabelled cholesterol during laboratory-simulated aerobic sewage treatment was studied by reverse phase radio-high performance liquid chromatography (rHPLC) and radio-gas chromatography (rGC) to examine the hypothesis that cholesterol could undergo A-ring aromatisation to form first, 19-norcholest-1,3,5(10)-trien-3-ol (NCT) and hence, by known bacterial pathways, the estrogenic steroid, estrone. The results showed that, to the contrary, much of the cholesterol (approx. 50% in 96 h) underwent rapid mineralisation to carbon dioxide, consistent with A-ring rupture (rather than aromatisation) and β-oxidation of the alkyl side chain as major transformation routes. Some polar (early-eluting) rHPLC products were observed, possibly steroidal conjugates and/or fatty acids. Among the minor metabolites detected by rGC and GC-mass spectrometry (GC-MS) were cholest-3,5-diene and a second cholestadiene isomer. However, since alkenes were unexpected in this rHPLC fraction, they may arise as artefacts from thermal decomposition of cholesteryl esters, indicating that some cholesterol esterification had also occurred. In the alcohol rHPLC fractions, cholestadienol was identified by GC and GC-MS but neither NCT or estrone were detected. This suggests that, at least under these simulated conditions, in situ aromatisation of cholesterol to NCT and formation of estrone from NCT were not major processes.

Quality assurance for PET gas production using the Cyclone 3D oxygen-15 generator

The production and quality control analysis of short-lived ( t 1/2=2.03 min) oxygen-15 labelled gases ([ 15O ]O 2, [ 15O ]CO, and [ 15O ]CO 2) from a Cyclone 3D accelerator for positron emission tomography (PET) in human subjects has been established and evaluated on a routine basis use at the MRC Cyclotron Unit and are here described. Contaminant levels have been identified and measured by radio-gas chromatography. The maximum levels allowed, measured as a percentage of total delivered activity (v/v), are 3.7% for total nitrogen oxides and 1% for [ 15O ]CO in [ 15O ]CO 2 and [ 15O ]CO 2 in [ 15O ]CO. By adjunction of a cryogenic or molecular sieve trap, radiopharmaceutical gases are routinely produced well within these specifications.

The biosynthesis of docosahexaenoic acid [22:6(n-3)] from linolenic acid in primary hepatocytes isolated from wild northern pike

Primary hepatocytes from wild northern pike Esox lucius were incubated with radiolabelled linolenic acid ([l-14C]-18:3(n-3)) to assess their ability to synthesize docosahexaenoic acid [22:6(n-3)]. The distribution of radioactivity in lipid classes and hepatocyte polyunsaturated fatty acids (PUFA) was measured over the time-course of 24h. The majority of radioactivity from [l-14C]-18:3(n-3) was recovered in hepatocyte triacylglycerols (TAG) and phosphatidylcholine (PC). The levels of radioactivity in TAG and in most of phospholipids, including PC, increased significantly over the incubation period. Radioactivity from [1-14C]-18:3(n-3) was recovered in several hepatocyte PUFA, including 22:6(n-3), and the Δ6 and Δ5-desaturation products 18:4(n-3) and 20:5(n-3). The presence of radioactivity in C24 (n-3) PUFA may be evidence that the biosynthesis of 22:6(n-3) in pike proceeds via a pathway independent of Δ4-desaturation. Analysis by radio gas chromatography revealed that radiolabelled 24:6(n-3) was present among the desaturation and elongation products of [l-14C]-18:3(n-3). The results establish that, under the in vitro conditions employed, pike hepatocytes are able to convert linolenic acid to 20:5(n-3) and 22:6(n-3).

The biosynthesis of docosahexaenoic acid [22:6(n-3)] from linolenic acid in primary hepatocytes isolated from wild northern pike.

Primary hepatocytes from wild northern pike Esox lucius were incubated with radiolabelled linolenic acid ([l-14 C]-18:3(n-3)) to assess their ability to synthesize docosahexaenoic acid [22:6(n-3)]. The distribution of radioactivity in lipid classes and hepatocyte polyunsaturated fatty acids (PUFA) was measured over the time-course of 24h. The majority of radioactivity from [l-14 C]-18:3(n-3) was recovered in hepatocyte triacylglycerols (TAG) and phosphatidylcholine (PC). The levels of radioactivity in TAG and in most of phospholipids, including PC, increased significantly over the incubation period. Radioactivity from [1-14 C]-18:3(n-3) was recovered in several hepatocyte PUFA, including 22:6(n-3), and the Δ6 and Δ5-desaturation products 18:4(n-3) and 20:5(n-3). The presence of radioactivity in C24 (n-3) PUFA may be evidence that the biosynthesis of 22:6(n-3) in pike proceeds via a pathway independent of Δ4-desaturation. Analysis by radio gas chromatography revealed that radiolabelled 24:6(n-3) was present among the desaturation and elongation products of [l-14 C]-18:3(n-3). The results establish that, under the in vitro conditions employed, pike hepatocytes are able to convert linolenic acid to 20:5(n-3) and 22:6(n-3).

Quantitative Analysis of Hydrogen Isotopes with a Mordenite Column at 173 K by Radio Gas Chromatography

Gas chromatography with a modified mordenite column was shown to yield a good hydrogen isotope separation at only 173 K. A linear relationship between peak height and concentration over several orders of magnitude was observed. From an extrapolation of the data it is concluded that all six hydrogen isotopes can be separated in only 13 min. Gas chromatographically separated radioactive hydrogens were detected with a small volume ionization chamber. The detection limit achieved for tritium was found to be about 0.3 ppm.

Characterization of Tritiated Hydrocarbon Species and Measurement of Their Concentrations in Air during Vacuum Pump Maintenance

When organic materials are exposed to elemental tritium, the hydrocarbons incorporate the tritium as a result of radiation induced reactions. The use of oil based vacuum pumps in tritium service gives rise to waste oil which contains, in addition to significant amounts of dissolved HT and HTO, a complex mixture of dissolved hydrocarbon species with a wide range of volatilities. The behavior of these species in the human body is not as well understood, and consequently, reliable dosimetry models only exist for the two most common tritiated species, HT and HTO. A quantitative radio-gas chromatography technique was developed to analyse the head space above waste vacuum pump oils. The technique was then applied at the Darlington Tritium Removal Facility (TRF) to assess the tritium exposure to personnel changing the oil from one of the high tritium cold boxes. 7 refs., 2 figs., 2 tabs.

Characteristics of the chemical forms of 11C, 13N, and 15O induced in air by the operation of a 100 MeV electron linear accelerator.

To characterize airborne radioactivity induced by the operation of high-energy accelerators, the fractions of aerosol and gaseous components, and the chemical forms of 11C, 13N, and 15O produced in the air of a target room of a 100 MeV electron linear accelerator were studied. Measurements of radioactivity using a particulate air sampling filter and a gas flow-through ionization chamber showed that more than 98% of 11C, 13N, and 15O were present as gaseous forms. Their chemical forms, detected by means of radio-gas chromatography, were 11C as CO2; 13N as N2 and NO; and 15O as O2 and NO. Machine operating conditions, which affect the compositions of the induced radionuclides and of their chemical forms, and the resulting effect on the estimation of internal doses are discussed.

Hydrocarbon biosynthesis in Triatoma infestans eggs.

Triatoma infestans eggs are shown to synthesize hydrocarbons. Radio-gas chromatography was used to demonstrate metabolism of [1-14C]propionate into precursor methyl-branched fatty acids and into methyl-branched hydrocarbons in T. infestans eggs. These reactions have not been demonstrated previously in insect eggs. An in vivo study showed that hydrocarbons are also transported to eggs by the hemolymph. Inhibition of hydrocarbon synthesis by sodium trichloroacetate (NaTCA) was correlated with reduced oviposition, reduced hatchability, and reduced insect survival. Scanning electron microscopy showed impoverishment of the eggs' epicuticular waxes following NaTCA treatment.

De novo biosynthesis of linoleic acid in two non-insect invertebrates: The land slug and the garden snail

The de novo biosynthesis of linoleic acid [18:2(n-6)], a fatty acid considered to be essential for most animals, was demonstrated in the land snail,Bulimulis alternatus mariae, and the garden slug,Arion circumscriptus. Radiolabeled acetate injected into the animals was incorporated into both 18:2 and 20:2, as demonstrated by radio-high performance liquid chromatography (radio-HPLC) and radio-gas chromatography (radio-GC). GC-mass spectrometry (GC-MS) of the methoxy derivatives of the 18:2 and 20:2 isolated from the snail showed that major isomers had the double bonds in the n-6,9 positions. Radio-gas-liquid chromatography (radio-GLC) of the ozonolysis products from the labeled dienoic fatty acid methyl esters showed that both ends of the molecules were labeled, confirming de novo synthesis. The production of linoleic acid by these animals suggests the capability to produce linoleic acid may be widespread in invertebrates.

Profiling of arachidonic acid metabolites in rabbit platelets by radio gas chromatography

A method for profiling arachidonic acid metabolites by radio gas chromatography (GC) is described. The incubation mixture of rabbit platelets with [14C]arachidonic acid was purified on a Sep-Pak C18 cartridge and derivatized with diazomethane, O-methylhydroxylamine and dimethyl-isopropylsilylimidazole. The recovery of total 14C-radioactivity was 93.1 +/- 7.2%. Loss of radioactivity during derivatization was negligible. Baseline separations for [14C]arachidonic acid and its metabolites were obtained in a single run within 45 min by GC using a synchronized accumulating radioisotope detector (GC/SARD). The recovery of radioactivity from the GC column was virtually 100%. The chemical structures of the metabolites were confirmed by GC/mass spectrometry; peaks of arachidonic acid metabolites were assigned by comparison of the methylene unit values with those of radioactive peaks in GC/SARD analyses. The intra-assay coefficients of variation in GC/SARD analyses were less than 10%. The method was used to map the profile of arachidonic acid metabolites formed by rabbit platelets in the presence of indomethacin, baicalein or glutathione.

Development of a chemical form discrimination system for 14C species in gaseous waste.

A new system was developed for discriminating the chemical form of 14C species in gaseous waste from nuclear facilities. The system consists of an air sample reservoir, a concentration apparatus, a radio-gas chromatograph, and microcomputers. An air sample, which is part of the gaseous effluent released from a stack of a facility, is automatically collected in the tank by trigger signals of stack monitors. After passing the air through a silica gel column, carbon species contained in it are recovered in an activated carbon-supported palladium trap held at -120 degrees C. The recovered species are swept with helium gas and analyzed by the radiogas chromatograph using an activated carbon column and gas-flow counters. Output signals of the gas-flow counters are continuously recorded by the microcomputer. By using this system, the chemical forms of 14C, such as CO, CO2, CH4, C2H4, C2H6, and C3H8 are identified and quantified. A detectable concentration of 0.3 mBq cm-3 for 14CO2 can be obtained. The system has been used to monitor the gaseous waste released from the radioisotope production laboratory and the Japan Research Reactor 2 at Japan Atomic Energy Research Institute.

On-line Tritium Process Gas Analysis by Laser Raman Spectroscopy at TSTA

Laser Raman spectroscopy has been applied to the on-line analysis of the operation of the cryogenic Isotope Separation System (ISS) at the Tritium Systems Test Assembly (TSTA). A flow-through cell was employed to permit near real-time observation of the dynamic response of the 3-column ISS. Accurate analysis of hydrogen isotopic mixtures may be made in less than 2 minutes. Full response to a change in the sampling point is achieved in approximately one minute. In this paper, response measurements are shown as well as static column profiles and dynamic response to induced parameter changes. Cross check of analysis was performed with radio-gas chromatography.

Formation of C21 bile acids from plant sterols in the rat.

Formation of bile acids from sitosterol in bile-fistulated female Wistar rats was studied with use of 4-14C-labeled sitosterol and sitosterol labeled with 3H in specific positions. The major part (about 75%) of the 14C radioactivity recovered as bile acids in bile after intravenous administration of [4-14C]sitosterol was found to be considerably more polar than cholic acid, and only trace amounts of radioactivity had chromatographic properties similar to those of cholic acid and chenodeoxycholic acid. It was shown that polar metabolites were formed by intermediate oxidation of the 3 beta-hydroxyl group (loss of 3H from 3 alpha-3H-labeled sitosterol) and that the most polar fraction did not contain a hydroxyl group at C7 (retention of 3H in 7 alpha,7 beta-3H2-labeled sitosterol). Furthermore, the polar metabolites had lost at least the terminal 6 or 7 carbon atoms of the side chain (loss of 3H from 22,23-3H2- and 24,28-3H2-labeled sitosterol). Experiments with 3H-labeled 7 alpha-hydroxysitosterol and 4-14C-labeled 26-hydroxysitosterol showed that none of these compounds was an efficient precursor to the polar metabolites. By analysis of purified most polar products of [4-14C] sitosterol by radio-gas chromatography and the same products of 7 alpha,7 beta-[2H2]sitosterol by combined gas chromatography-mass spectrometry, two major metabolites could be identified as C21 bile acids. One metabolite had three hydroxyl groups (3 alpha, 15, and unknown), and one had two hydroxyl groups (3 alpha, 15) and one keto group. Considerably less C21 bile acids were formed from [4-14C]sitosterol in male than in female Wistar rats. The C21 bile acids formed in male rats did not contain a 15-hydroxyl group. Conversion of a [4-14C]sitosterol into C21 bile acids did also occur in adrenalectomized and ovariectomized rats, indicating that endocrine tissues are not involved. Experiments with isolated perfused liver gave direct evidence that the overall conversion of sitosterol into C21 bile acids occurs in this organ. Intravenously injected 7 alpha,7 beta-3H-labeled campesterol gave a product pattern identical to that of 4-14C-labeled sitosterol. Possible mechanisms for hepatic conversion of sitosterol and campesterol into C21 bile acids are discussed.

Use of radio-gas chromatography and tritium label to optimize derivatization procedures

A sensitive determination of gas chromatographic peak yields by a new radio-gas chromatograph equipped with a synchronized accumulating radioisotope detector is described. Peak yields could be easily determined by the radio-gas chromatograph using [ 3H]hexadecane or [ 3H]androstenedione as a standard substance. The usefulness of the determination of peak yields was demonstrated by optimizing the derivatization conditions of 6-keto-prostaglandin F 1 α .