Oxidative decarboxylation of benzoic acid by peroxyl radicals.

A chemical model based on the thermal decomposition of AAPH (2,2'-azobis(2-amidinopropane) dihydrochloride is used for the production of peroxyl radicals. Peroxyl radicals induces the decarboxylation of [7-13C]benzoic acid and the production of 13CO2, which is measured by gas chromatography-isotope ratio mass spectrometry (GC-IRMS). The decarboxylation depends on temperature, AAPH, and benzoic acid concentrations. The decarboxylation also depends on the presence of oxygen. Electron spin resonance studies are performed to confirm the presence of peroxyl radicals under oxygen and of carbon-centered radicals in the absence of oxygen. Decarboxylation rates are measured in the presence of various antioxidants: ascorbate, dimethylsulfoxide, mannitol, and uric acid. It turns out that the decarboxylation is inhibited by each of these antioxidants. The ratio of decarboxylation rates, with and without the antioxidant, varies linearly with the antioxidant concentration. HPLC and GC-MS analyses of reaction products between benzoic acid and AAPH-derived radicals do not detect the presence of radical substitution products on the aromatic ring or the products derived from benzoic acid. There is no doubt that GC-IRMS is a powerful technique to investigate the effects of peroxyl radicals on benzoic acid. In addition, it is possible to follow the degradation of 13C-labeled chemical targets exposed to peroxyl radicals through the production of 13CO2.

13C appearance in plasma glucose and breath CO2 during feeding with naturally 13C-enriched starchy food in normal humans.

We have used a recently developed technique (isotope-ratio mass spectrometer) to measure 13C appearance in plasma glucose and breath CO2 in eight normal subjects during feeding with naturally 13C-enriched starch. 13C in CO2 and plasma glucose, metabolites and insulin concentrations, carbohydrates, and lipid oxidation were measured after ingestion of 76 g glucose equivalent of crackers, pasta, or polenta. 13C in plasma glucose displays a very different pattern from plasma glucose concentration. It increases steadily for 90 min before reaching a plateau for approximately 2 h and slowly declines during the last 2 h of the study. No significant difference was observed with the three different starchy foods tested although plasma glucose tended to be higher during feeding with polenta. In summary measurement of 13C in plasma glucose during feeding with naturally 13C-labeled carbohydrates yields new insight in the study of carbohydrate bioavailability in humans.

The 4-O-methylation pathway of catecholamines: search for an in vivo production of 3-hydroxy,4-methoxyphenylglycol (iso-MHPG).

The existence of 3-hydroxy,4-methoxyphenylglycol in biological fluids has been investigated. 4-Hydroxy,3-methoxy-phenylglycol (MHPG) and 3-hydroxy,4-methoxyphenylglycol (iso-MHPG) have been separated from each other by gas-liquid chromatography of their heptafluorobutyryl derivatives, using an electron capture detector procedure. Urines of normal subjects, of patients with secreting nervous tissue tumors and human cerebrospinal fluids were analyzed. Iso-MHPG was never detected, with the exception of trace amounts in one urinary sample from a patient with a peculiarly high excretion of MHPG. It is concluded that norepinephrine is probably not 4-O-methylated in vivo, contrary to dopamine which is known to lead, at least to a small extent, to 4-O-methylated metabolites.

Continuous monitoring of 13C-aminopyrine metabolism in rats: effects of cold exposure and noradrenaline.

A system was developed to allow constant monitoring of hepatic cytochrome P450 activity in awake and unrestrained rats. A continuous 13C-aminopyrine perfusion was performed, and breath samples obtained for endogenous CO2 production and 13C measurements, to calculate 13C O2 production due to aminopyrine demthylation. Increasing doses of 13C-aminopyrine produced a hyperbolic increase of expired 13CO2, compatible with an in vivo measurement of enzymatic activity. Acute-cold exposure of the rats during 13C-aminopyrine perfusion produced a two-fold increase of endogenous CO2 production, together with a 27% increased 13C-aminopyrine metabolism (p<0.05 vs basal conditions). In contrast, noradrenaline (20 microg/kg BW/min), despite a similar effect on energy expenditure, did not significantly change 13C-aminopyrine metabolism. Acute-cold exposure is known to stimulate both adrenal catecholamine secretion and the sympathetic nervous system. The observed difference in 13C-aminopyrine demthylation during cold exposure and nonadrenaline perfusion, therefore, could be due to a more specific effect of adrenal catecholamines on liver aminopyrine metabolism. These results suggest the possibility of prolonged in vivo monitoring of liver metabolism pathways such as aminopyrine demethylation, thus allowing the study of drug acute interactions with cytochrome P450 system.

Action of methotrexate on cytochrome P-450 monooxygenases in rats. Study performed with [13C]-aminopyrine micro breath test.

The aim of this work was to study the action of methotrexate on the cytochrome P-450 hepatic monooxygenases and to follow the evolution of the metabolic processes with time, after oral and intra-peritoneal administration of these drugs to rats. In order to perform this study, we used a new technique the [13C]-aminopyrine breath test (ABT). At the end of the in vivo study, the rats were sacrificed and the cytochrome P-450 concentration as well as microsomal enzymatic activities of aminopyrine N-demethylase, 7-ethoxyresorufin desalkylase, 7-ethoxycoumarin desalkylase, 7-pentoxyresorufin desalkylase were determined. A histological study of the liver was also carried out. Finally, haematological and biochemical parameters were also determined. In this study, 3 series of 6 rats were used: a control group, a group receiving 70 mg/kg of methotrexate by intra-peritoneal route, and a group of rats treated orally with 1 mg/kg of methotrexate every 2 days. For each observation, the variation of the ABT scores and of the cytochrome P-450 amounts as well as the microsomal aminopyrine N-demethylase activity were in good agreement.

On-line measurements of 13C enrichments in rat breath. Non-invasive method for in vivo study of drug enzymatic induction.

A differential kinetic study of 13CO2 enrichment of breath after the intake of specific 13C-labelled substrates and co-administration of a drug allows the drug's ability for enzyme induction to be evaluated in vivo. A method and a gas chromatograph-isotope ratio mass spectrometer device for on-line measurements of 13CO2 enrichment in the breath of small animals are described. This system allows on-line breath sample collection from a metabolic cage, purification by gas chromatography, determination of CO2 by thermal conductivity detection and measurement of 13CO2 enrichment by isotope ratio mass spectrometry. Two protocols for phenobarbital-inducible P450 and 3-methylcholanthrene-inducible P1-450 isoenzymes are described.

In vivo metabolism of aminopyrine by the larvae of the helminth Heligmosomoides polygyrus.

The in vivo N-dealkylation of [13C-2]-labeled aminopyrine by the L1-L2 larvae of Heligmosomoides polygyrus was demonstrated by the use of a sensitive gas chromatography-mass spectrometry method. This is the first evidence for the possible existence of a cytochrome P-450-dependent activity in helminths.

Reverse isotope dilution analysis of 13CO2 using gas chromatography/isotope ratio mass spectrometry: application to the quantitative determination of 13CO2 released by human polymorphonuclear leukocytes.

We propose a new method for the quantitative determination of carbon dioxide released by a biological microgenerator: a suspension of human polymorphonuclear leukocytes (PMNL). This method is based on the reverse isotope dilution analysis by gas chromatography/isotope ratio mass spectrometry of 13CO2 released by PMNL in a controlled isotope abundance atmosphere containing 3% CO2. 13CO2 release is effective after PMNL stimulation in the presence of [13C]glucose, labeled on positions 1, 2 or 6. The validation of this method is carried out by the measurement of the isotope ratio 13CO2/12CO2 using known amounts of [13C]sodium hydrogen carbonate and the comparison with theoretical isotope abundances derived from various CO2 equilibria. Complete release of CO2 is achieved by the acidification of the medium. This method requires only few cells, displays high sensitivity and specificity and can be applied to the analysis of large series of samples using an automatic sample injector. In addition, this method can also be applied to other types of biological microgenerators of carbon dioxide.

Evaluation of hydroxyl radicals production using 13CO2 gas chromatography-isotope ratio mass spectrometry.

A gas chromatography-isotope ratio mass spectrometry (GC-IRMS) technique for detecting the production of hydroxyl radicals is described. The decarboxylation of [7-13COOH]benzoic acid in the presence of a hydroxyl radicals source (a mixture of porphyrin and hydrogen peroxide) was followed by direct measurement of the 13CO2/12CO2 isotopic ratio. The production of hydroxyl radicals by the mixtures of porphyrin-hydrogen peroxide was proved by comparative study with electron spin resonance spectrometry and high-performance liquid chromatography analysis of hydroxylation products of benzoic acid. The water-soluble radical scavengers methanol, mannitol, and dimethyl sulfoxide led to the inhibition of 13CO2 production from [7-13COOH]benzoic acid. In contrast, high concentrations of the antioxidant ascorbate strongly increased [7-13COOH]benzoic acid decarboxylation. Finally, the use of anaerobic conditions showed that decarboxylation was independent of the presence of oxygen. The absence of the effect of superoxide dismutase could exclude a possible effect of the superoxide ion. This nonradioactive technique offers many advantages compared to the well-established method for detecting hydroxyl radicals based on the decarboxylation of [7-14COOH]benzoic acid. It is rapid and easy to perform as a simple tube test and is highly reliable for detecting hydroxyl radicals. This method provides an on-line analysis of carbon dioxide compared to the radiochemical method. In addition, 13CO2-enrichment measurements led to easy kinetic studies with high sensitivity and semiquantitative determinations.

Positional isotopic analysis of 13C-labelled glucose by mass spectrometry: applications to the study of gluconeogenesis in liver cells.

The aim of the present investigation was to ascertain whether mass spectrometric analysis of glucose allows determination in small samples (0.01 nmol) of the sites and the extent of labelling of glucose produced by isolated liver cells from various gluconeogenic labelled precursors. The electron impact spectrum of the methyloxime pentatrimethylsilyl derivative of natural glucose affords fragment ions retaining specific carbon atoms, i.e. 1-2 (m/z 160), 1-2-3 (m/z 262), 3-4-5-6 (m/z 319), 4-5-6 (m/z 217), 5-6 (m/z 205), 6 (m/z 103). The mass fragmentography analysis of the same derivative of commercially available labelled glucose molecules (1-13C, 6-13C, 2-2H, 3-2H, 6,6-2H2) permitted evaluation of the degree of specificity of these fragment ions, and development of a calculation method for isotope incorporation. Using this methodology we found that incubation of hepatocytes with (2-13C)glycerol, (1,3-13C)glycerol or NaH13CO3 plus pyruvate or lactate produced (2,5-13C)glucose, (1,3,4,6-13C) glucose or (3,4-13C)glucose, respectively. The extent of labelling was measurable on individual carbon of the glucose molecule except for carbon 1. The lowest enrichment detectable on carbon 1-3 or 3 was found to be 0.5%. In conclusion, gas chromatography mass spectrometry is a reliable method for positional isotopic analysis of 13C-labelled glucose, and appears useful in the study of the gluconeogenic pathway.

Use of a new gas chromatograph isotope ratio mass spectrometer to trace exogenous 13C labelled glucose at a very low level of enrichment in man.

The use of 13C labelled glucose in human metabolic studies has been limited by the high cost of the tracer and the problems of measuring low 13C isotopic abundance in plasma glucose. In the present work we describe a new gas chromatograph-isotope ratio mass spectrometer allowing the measurement of a 0.001 atom % increase in 13C abundance over baseline, on a nanomole glucose sample. Studies were performed in rats and in human subjects. The rate of glucose appearance in 24 h fasted rats using D-[1-13C] glucose as tracer and analysed by this new method was found to be 10.4 +/- 0.7 mg.kg-1.min-1, a value 21% lower than that found using D-[6,6-2H2] glucose as tracer (13.1 +/- 1.1 mg.kg-1.min-1) analysed by classic gas chromatography-mass spectrometry. The new method was also used to trace, in combination with D-[6,6 2H2] glucose, the metabolic fate in human subjects of two oral glucose loads (0.5 g.kg.-1,1 g.kg.-1) labelled with 0.1% D-[U-13C] glucose. During the six hours following the glucose load, it was found that total glucose appearance was 0.97 +/- 0.04 g.kg.-1 and 1.2 +/- 0.04 g.kg.-1, exogenous glucose appearance was 0.51 +/- 0.02 g.kg.-1 and 0.84 +/- 0.04 g.kg.-1, endogenous glucose production was 0.44 +/- 0.04 g.kg.-1 and 0.35 +/- 0.06 g.kg.-1 after the 0.5 and 1 g.kg.-1 load respectively. These values are similar to those reported using glucose labelled with radioactive isotopes.(ABSTRACT TRUNCATED AT 250 WORDS)