[Validation of pharmaceutical quality of a [6,6-(2)H(2)]-glucose parenteral solution used for insulin-resistance measurement during human clinical trials]. / Validation de la qualité pharmaceutique de préparations de [6,6-(2)H(2)]-glucose en solution aqueuse administrées par voie parentérale pour la mesure de l'insulino-résistance dans le cadre d'essais cliniques.

INTRODUCTION: Deuterated glucose ([6,6-(2)H(2)]-glucose) is a stable isotopic tracer administered parenterally in healthy volunteers, obese or diabetic patients in clinical trial to study glucose metabolism during euglycemic hyperinsulinemic clamps. In accordance with the Health Authorities on drug safety, we evaluated the pharmaceutical quality of this preparation for biomedical research with a stability study. METHODS: After pharmaceutical qualification of the raw material, the [6,6-(2)H(2)]-glucose was dissolved in water for injection, then sterile, filtered under positive pressure of nitrogen and then autoclaved. Two batch products (500mg/10mL and 2g/15mL) were sampled to evaluate glucose alteration, isotope shift, limpidity, apyrogenicity and sterility at regular intervals for 2 years. Deuterated glucose solutions were stored in the dark, at +2°C+8°C, in type II glass bottles. RESULTS: Neither significant decrease of glucose concentration nor pH variation were observed for 2 years. The 5-hydroxymethylfurfural concentration was below the human harmful levels, attesting a non-generation of metabolites during autoclaving. Isotopic enrichment higher than 99% reflected the stability of deuterated label on the 6-carbon of glucose molecules. The non-visible particle concentration below the minimal permissible concentration tolerated by the European Pharmacopoeia and the absence of bacterial endotoxin and bacterial growth attested limpidity, apyrogenicity and sterility of the [6,6-(2)H(2)]-glucose solutions. CONCLUSION: After the 2-year study, 500mg/10mL and 2g/15mL deuterated glucose solutions stored in the dark at +2°C+8°C were stable in aqueous solution, allowing to ensure safety administration for human clinical trials using euglycemic hyperinsulinemic clamps.

Nutritional intervention to reduce the n-6/n-3 fatty acid ratio increases adiponectin concentration and fatty acid oxidation in healthy subjects.

BACKGROUND/OBJECTIVES: Consumption of n-3 polyunsaturated fatty acids (PUFA) has a favourable impact on inflammation and cardiovascular disease. However, the Western diet is characterized by a low n-3 PUFA intake and an imbalance in the n-6/n-3 PUFA ratio. Study the effect 10-week of diet modification to decrease the n-6/n-3 PUFA ratio on cardiovascular risk factors and resting energy expenditure. SUBJECTS AND METHODS: Ten-week dietary intervention in 17 healthy subjects. Dietary intake, euglycemic hyperinsulinemic clamp, indirect calorimetry, lipid profile, hormones, inflammatory markers and erythrocyte membrane fatty acid composition were recorded before and at the end of the intervention. Comparisons are between baseline and post-treatment levels. RESULTS: Dietary records of the linoleic acid/alpha-linolenic acid ratio (baseline: 32.2 (s.d. 3.7) vs post-intervention: 2.2 (s.d. 0.1), P<0.0001) and erythrocyte membrane fatty acid composition reflected good compliance. Dietary intervention was associated with significant reductions in TNF-alpha (baseline: 2.2 (s.d. 0.3), post-intervention: 1.5 (s.d. 0.3) pg/ml, P=0.01) and low-density lipoprotein-cholesterol (baseline: 2.5 (s.d. 0.2), post-intervention: 2.3 (s.d. 0.1) mmol/l, P=0.03) and increased adiponectin (baseline: 6.5 (s.d. 0.7), post-intervention: 7.6 (s.d. 0.6) microg/ml, P=0.02). Fasting lipid oxidation was increased (baseline: 0.7 (s.d. 0.1), post-intervention: 0.9 (s.d. 0.1) mg/kg x min, P=0.01), whereas glucose oxidation decreased in both fasting (baseline: 1.6 (s.d. 0.1), post-intervention: 1.3 (s.d. 0.1) mg/kg x min, P=0.02) and hyperinsulinaemic conditions (baseline: 3.6 (s.d. 0.1), post-intervention: 3.3 (s.d. 0.1) mg/kg x min, P=0.04). Insulin sensitivity was not affected by the intervention. CONCLUSION: A decreased n-6/n-3 PUFA ratio can be achieved with simple dietary counselling, resulting in multiple, potentially favourable effects on the metabolic and inflammatory profiles.

The effects of rosiglitazone on fatty acid and triglyceride metabolism in type 2 diabetes.

AIMS/HYPOTHESIS: We investigated the effects of rosiglitazone on NEFA and triglyceride metabolism in type 2 diabetes. METHODS: In a double-blind, placebo-controlled, cross-over study of rosiglitazone in diet-treated type 2 diabetic subjects, we measured arteriovenous differences and tissue blood flow in forearm muscle and subcutaneous abdominal adipose tissue, used stable isotope techniques, and analysed gene expression. Responses to a mixed meal containing [1,1,1-(13)C]tripalmitin were assessed. RESULTS: Rosiglitazone induced insulin sensitisation without altering fasting NEFA concentrations (-6.6%, p=0.16). Postprandial NEFA concentrations were lowered by rosiglitazone compared with placebo (-21%, p=0.04). Adipose tissue NEFA release was not decreased in the fasting state by rosiglitazone treatment (+24%, p=0.17) and was associated with an increased fasting hormone-sensitive lipase rate of action (+118%, p=0.01). Postprandial triglyceride concentrations were decreased by rosiglitazone treatment (-26%, p<0.01) despite unchanged fasting concentrations. Rosiglitazone did not change concentrations of triglyceride-rich lipoprotein remnants. Adipose tissue blood flow increased with rosiglitazone (+32%, p=0.03). Postprandial triglyceride [(13)C]palmitic acid concentrations were unchanged, whilst NEFA [(13)C]palmitic acid concentrations were decreased (p=0.04). In muscle, hexokinase II mRNA expression was increased by rosiglitazone (+166%, p=0.001) whilst the expression of genes involved in insulin signalling was unchanged. Adipose tissue expression of FABP4, LPL and FAT/CD36 was increased. CONCLUSIONS/INTERPRETATION: Rosiglitazone decreases postprandial NEFA and triglyceride concentrations. This may represent decreased spillover of NEFAs from adipose tissue depots. Decreased delivery of NEFAs to the liver may lead to lowered postprandial triglyceride concentrations. Upregulation of hexokinase II expression in muscle may contribute to insulin sensitisation by rosiglitazone.

Continuous flow isotope ratio mass spectrometry for the measurement of nanomole amounts of (13)CO(2) by a reverse isotope dilution method.

A simple method for the determination of nanomole amounts of (13)CO(2) generated from an in vitro reaction is reported. The incubation medium contains a known amount of unlabeled sodium bicarbonate and the gaseous (13)CO(2) enriches the atmosphere upon which a measurement of the isotopic enrichment ((13)CO(2)/(12)CO(2)) is made corresponding to a reverse isotope dilution. The quantification of the (13)CO(2) was performed by gas chromatography/isotope ratio mass spectrometry. This assay was validated in terms of linearity, accuracy and precision using three different substrates which produce (13)CO(2) either by enzymatic reaction [(13)C]urea, sodium [(13)C]formate) or by chemical reaction (sodium [(13)C]bicarbonate). Four calibration curves were tested for each (13)C-labeled substrate, allowing the quantification of (13)CO(2) from 25 pmol to 150 nmol. The dynamics of the assay were obtained as a function of the quantity of unlabeled sodium bicarbonate added to each sample.

Influence of dietary fat on postprandial glucose metabolism (exogenous and endogenous) using intrinsically (13)C-enriched durum wheat.

The present study evaluates the influence of different amounts of fat added to starch on postprandial glucose metabolism (exogenous and endogenous). Nine women (24 (se 2) years old, BMI 20.4 (se 0.7) kg/m(2)) ingested 1 week apart 75 g glucose equivalent of (13)C-labelled starch in the form of pasta without (low fat; LF) or with 15 (medium fat; MF) or 40 (high fat; HF) g sunflower oil. During the 7 h following meal consumption, plasma glucose, non-esterified fatty acids, triacylglycerols (TG) and insulin concentrations, and endogenous (using [6,6-(2)H(2)]glucose) and exogenous glucose turnover were determined. With MF and HF meals, a lower postprandial glucose peak was observed, but with a secondary recovery. A decrease in exogenous glucose appearance explained lower glycaemia in HF. At 4 h after the HF meal the insulin, insulin:glucose and postprandial blood TG were higher than those measured after the LF and MF meals. Despite higher insulinaemia, total glucose disappearance was similar and endogenous glucose production was suppressed less than after the LF and MF meals, suggesting insulin resistance. Thus, the addition of a large amount of fat appears to be unfavourable to glucose metabolism because it leads to a feature of insulin resistance. On the contrary, the MF meal did not have these adverse effects, but it was able to decrease the initial glycaemic peak.

The regulation of uncoupling protein-2 gene expression by omega-6 polyunsaturated fatty acids in human skeletal muscle cells involves multiple pathways, including the nuclear receptor peroxisome proliferator-activated receptor beta.

Fatty acids have been postulated to regulate uncoupling protein (UCP) gene expression in skeletal muscle in vivo. We have identified, at least in part, the mechanism by which polyunsaturated fatty acids increase UCP-2 expression in primary culture of human muscle cells. omega-6 fatty acids and arachidonic acid induced a 3-fold rise in UCP-2 mRNA levels possibly through transcriptional activation. This effect was prevented by indomethacin and mimicked by prostaglandin (PG) E(2) and carbaprostacyclin PGI(2), consistent with a cyclooxygenase-mediated process. Incubation of myotubes for 6 h with 100 micrometer arachidonic acid resulted in a 150-fold increase in PGE(2) and a 15-fold increase in PGI(2) in the culture medium. Consistent with a role of cAMP and protein kinase A, both prostaglandins induced a marked accumulation of cAMP in human myotubes, and forskolin reproduced the effect of arachidonic acid on UCP-2 mRNA expression. Inhibition of protein kinase A with H-89 suppressed the effect of PGE(2), whereas cPGI(2) and arachidonic acid were still able to increase ucp-2 gene expression, suggesting additional mechanisms. We found, however, that the MAP kinase pathway was not involved. Prostaglandins, particularly PGI(2), are potent activators of the peroxisome proliferator-activated receptors. A specific agonist of peroxisome proliferator-activated receptor (PPAR) beta (L165041) increased UCP-2 mRNA levels in myotubes, whereas activation of PPARalpha or PPARgamma was ineffective. These results suggest thus that ucp-2 gene expression is regulated by omega-6 fatty acids in human muscle cells through mechanisms involving at least protein kinase A and the nuclear receptor PPARbeta.

Detection of new propofol metabolites in human urine using gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry techniques.

Using hyphenated analytical techniques, gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), a study on minor propofol metabolites in human urine was conducted. These techniques allowed identification of two new phase I metabolites (2-(omega-propanol)-6-isopropylphenol and 2-(omega-propanol)-6-isopropyl-1,4-quinol). In addition, their four corresponding conjugates (three glucuronides and one sulphate) were detected. Thus in human urine at least eight conjugate metabolites are produced, derived from four different aglycones (propofol; 2, 6-diisopropyl-1,4-quinol; 2-(omega-propanol)-6-isopropylphenol and 2-(omega-propanol)-6-isopropyl-1,4-quinol).

Possible involvement of multiple human cytochrome P450 isoforms in the liver metabolism of propofol.

Previous studies of propofol (2,6-diisopropylphenol) pharmacology have shown that this widely used anaesthetic drug is extensively cleared from the body by conjugation of the parent molecule or its quinol metabolite. On the basis of potential influence of propofol on the metabolism of co-administered agents, many investigators have evaluated the effects of propofol on cytochrome P450 (CYP) activities. CYP isoforms involved in propofol metabolism are not defined. In this study, our objective was to elucidate further the CYP isoforms responsible for the hydroxylation of propofol. Using microsomes from 12 different human livers, we investigated CYP isoforms involved in propofol hydroxylase activity, using selective chemical inhibitors of CYP isoforms, correlation with immunoquantified specific CYP isoform content, immunoinhibition, and 11 functionally active human CYP isoforms expressed in a heterologous system (yeast and human B-lymphoblastoid cells). We found a low variability in the production of the hydroxylated metabolite of propofol, 2,6-diisopropyl-1,4-quinol. This activity was mediated by CYP and followed Michaelis-Menten kinetics with apparent K(M) and Vmax values of 18 microM (95% Cl 15.1-20.1) and 2.6 nmol min-1 mg-1 (95% Cl 2.45-2.68) respectively. Part of the propofol hydroxylase activity was mediated by CYP2C9 in human liver, especially at low substrate concentration. Moreover, propofol was likely to be metabolized by additional isoforms such as CYP2A6, 2C8, 2C18, 2C19 and 1A2, especially when substrate concentrations are high. This low specificity among CYP isoforms may contribute to the low interindividual variability (two-fold) and may contribute to the low level of metabolic drug interactions observed with propofol.

Decarboxylation of [1-(13)C]leucine by hydroxyl radicals.

The decarboxylation of [1-13C]leucine by hydroxyl radicals was studied by using gas chromatography-isotope ratio mass spectrometry (GC-IRMS) to follow the production of 13CO2. A Fenton reaction between a (Fe2+)-porphyrin and hydrogen peroxide under aerobic conditions yielded hydroxyl radicals. The decarboxylation rates (VLeu) measured by GC-IRMS were dependent on [1-13C]leucine, porphyrin and hydrogen peroxide concentrations. The 13CO2 production was also dependent on bicarbonate or carbon dioxide added in the reaction medium. Bicarbonate facilitated 13CO2 production, whereas carbon dioxide decreased 13CO2 production. Proton effects on some decarboxylation intermediates could explain bicarbonate or carbon dioxide effects. No effect on the decarboxylation rates was observed in the presence of the classical hydroxyl radicals scavengers dimethyl sulfoxide, mannitol, and uric acid. By contrast, a competitive effect with a strong decrease of the decarboxylation rates was observed in the presence of various amino acids: unlabeled leucine, valine, phenylalanine, cysteine, lysine, and histidine. Two reaction products, methyl-4 oxo-2 pentanoate and methyl-3 butanoate were identified by gas chromatography-mass spectrometry in comparison with standards. The present results suggest that [1-13C]leucine can participate to the coordination sphere of (Fe2+)-porphyrin, with a caged process of the hydroxyl radicals which cannot get out of the coordination sphere.

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.

Possible involvement of multiple cytochrome P450S in fentanyl and sufentanil metabolism as opposed to alfentanil.

Fentanyl, sufentanil, and alfentanil are commonly used as opioid analgesics. Alfentanil clearance has previously been shown to exhibit an important interindividual variability, which was not observed for fentanyl or sufentanil. Differences in pharmacokinetic parameters of alfentanil have previously been associated with the wide distribution of CYP3A4, the only known hepatic cytochrome P450 monooxygenase (CYP) involved in the conversion of alfentanil to noralfentanil. Little is known about the involvement of CYP enzymes in the oxidative metabolism of fentanyl and sufentanil. Microsomes prepared from different human liver samples were compared for their abilities to metabolize fentanyl, sufentanil and alfentanil, and it was found that disappearance of the three substrates was well correlated with immunoreactive CYP3A4 contents but not with other CYPs, including CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6 and CYP2E1. Specific known inhibitors of CYP enzymes gave similar results, whereas the use of recombinant human CYP enzymes expressed in yeast provided information about the possible involvement of other CYPs than CYP3A4 in the biotransformation of fentanyl and sufentanil. The possible in vivo interaction of fentanyl and sufentanil with other drugs catalyzed by CYP3A4 is also discussed.

Gas chromatographic-mass spectrometry and gas chromatographic-Fourier transform infrared spectroscopy assay for the simultaneous identification of fentanyl metabolites.

Fentanyl, a synthetic opioid, undergoes important biotransformation to several metabolites. A gas chromatographic-mass spectrometric assay was applied for the simultaneous analysis of fentanyl and its major metabolites in biological samples. The identification of different metabolites was performed by gas chromatography-mass spectrometry (electronic impact and chemical ionisation modes) and gas chromatography-Fourier transform infrared spectroscopy. In the present study, rat and human microsomes incubation mixtures and human urines were analysed. In vitro formation of already known fentanyl metabolites was confirmed. The presence of metabolites not previously detected in human urine is described.

Deuterium isotope effects on caffeine metabolism.

The aim of this work was to study the influence of labelling on caffeine metabolism according to the incubation mode. It has been observed that labelling induces an isotopic effect on metabolisation speed: apparent half-lives are systematically increased. Moreover, isotopic effects are in agreement with those previously observed for retention times. Qualitatively, caffeine labelling systematically induces metabolic variations to the detriment of the metabolic pathway that induces the loss of the trideuteromethyl group. Caffeine accumulation and the release of the main metabolites have been studied with respect to isotopic effects on crossing the cell membrane. It has been demonstrated that, in most of the cases, accumulation decreased and that either metabolite release was increased or no isotopic effect was observed.

Isotopic effects on retention times of caffeine and its metabolites 1,3,7-trimethyluric acid, theophylline, theobromine and paraxanthine.

Physicochemical parameters that influence gas chromatographic separation are numerous. Consequently, isotope labelling, because it modifies physicochemical properties, can induce isotopic effects on retention time. Caffeine has been chosen to study this influence because as itself and its metabolites, it allows the preparation of different methylxanthine isotopomers and thus is one of the best models to study isotopic effects induced by stable isotope labelling. Using a caffeine molecule labelled with deuterium at different positions and rat hepatocytes to obtain metabolites, it was possible to study the influence of labelling on retention time [(14% cyanopropylphenyl)methylpolysiloxane] and to point out the role of each labelled site. It appears that isotopic effects induced by the labelling depend not only on the number of labelling atoms but also on whether this labelling is at position 1, 3 or 7 and, consequently, on the role of the labelled site on the function of the molecule.

Comparison between gas chromatography-atomic emission detection and gas chromatography-mass spectrometry for the assay of propofol.

Quantitation by gas chromatography-atomic emission detection (GC-AED) is based on the intensity of the signal measured at a wavelength characteristic of an element, after atomisation by the plasma. This response depends only on the number of atoms of this element present in the molecule under investigation, and is independent of the structure of the molecule. This technique was used for the assay of propofol, and the estimation of its two metabolites, after calibration with standard solutions of pure propofol. The results were compared with those obtained by gas chromatography-mass spectrometry (GC-MS). Propofol was quantified with higher precision and accuracy by GC-AED than by GC-MS which exhibited larger residual values. Concentration assessment for two metabolites showed a better agreement with the theoretical value by GC-AED since the response depends only on the number of carbon atoms in each molecule.

Gas chromatographic-mass spectrometric method to characterise the transfer of dietary odorous compounds into plasma and milk.

The flavours contained in a mammalian mother's milk can exert a marked influence on her offspring's proximate suckling behaviour and later preferences. The aim of this study was to establish a reliable analytical procedure to characterise the mammary transfer of selected volatile constituents of maternal food from non-pregnant and recently parturient ewes. Six known volatile compounds, most representative of cumin aroma (alpha-pinene, gamma-terpinene, cuminaldehyde, p-cymene, limonene and cineole), were traced in the blood and milk of ewes fed with cumin seeds, using liquid-liquid extraction combined with gas chromatography-specific ion monitoring mass spectrometry. Among the six cumin odour markers, only one, p-cymene, was transferred in quantifiable amounts into the venous plasma. The other cumin markers could only be detected as traces corresponding to amounts lower that the limit of quantification. In milk, four of the cumin markers could be detected, and two of these were quantified.

Quantitation of propofol in whole blood by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric assay, using selected-ion monitoring (GC-MS-SIM) with thymol as internal standard, was developed for quantitating propofol, an intravenous anaesthetic. The method described is rapid and sensitive for the determination of propofol in whole blood. The sensitivity of the present method is 10 ng/ml. The recovery of propofol added to human whole blood in the concentration range 10-10,000 ng/ml ranged between 95 and 100%. A single extraction procedure was used with chloroform-ethyl acetate. The assay allowed the detection of two metabolites formed during propofol metabolism: 2,6-diisopropyl-1,4-quinone and 2,6-diisopropyl-1,4-quinol.

Effects of capsaicinoids on oxidative metabolism of caffeine in isolated rat hepatocytes.

The metabolism of caffeine was studied in isolated rat hepatocytes, in the absence and presence of capsaicinoids. Caffeine and four primary metabolite fractions were identified by high performance liquid chromatography: 1,7-dimethylxanthine, 3,7-dimethylxanthine, 1,3-dimethylxanthine and 1,3,7-trimethyluric acid. The incubation with the lowest concentrations (0.1 and 1 microM) of capsaicinoids (natural extract, capsaicin, dihydrocapsaicin) showed a stimulatory effect on caffeine metabolism, which was further enhanced with capsaicin. At 10 microM, capsaicin stimulated the two pathways of metabolism of caffeine (N-demethylation and C-8 oxidation). In contrast, dihydrocapsaicin and the natural extract seem to inhibit the N-demethylation pathways without affecting the C-8 oxidation route. The inhibitory activity on the N-demethylation pathways and especially the N-7 demethylation pathway was pronounced at the first 30 min of incubation. These results suggest that the two pathways (N-demethylation and C-8 oxidation) are mediated by different isozymes of cytochromes P-450. This is in agreement with recent findings.

Detection of 13C labelled compounds by gas chromatography coupled to atomic emission detection--application to caffeine metabolites.

This paper illustrates the use of gas chromatography coupled to atomic emission detection (GC-AED) and of the stable isotope tracer 13C for the determination of drug metabolites. After administration of a parent drug labelled with 13C and extraction of the metabolites from the biological fluids, a 13C chromatographic profile is determined using the specific detection of the 13C atomic emission and subtraction of the 13C natural abundance. Thus, only the compounds which are metabolites with a 13C enrichment over the natural abundance are detected. [1,3,7 trimethyl-13C3]xanthine which is extensively metabolized by the liver is used as an example.

Quantitation of trihexyphenidyl from plasma using a mass-selective detector and electron-impact ionization.

A method is described for the measurement of plasma concentrations of trihexyphenidyl, an anti-parkinsonian drug. The drug was extracted from human plasma samples. Then, gas chromatography-mass spectrometry with electron-impact ionization and selected-ion monitoring allowed the specific quantitation of trihexyphenidyl, with bupivacaine used as an internal standard. Linear calibration curves were obtained in the concentration range 5-100 ng/ml. Precision and accuracy were found acceptable for quantitation during pharmacokinetic trials of the drug. This method has been successfully applied to bioavailability studies after Parkinane and Artane administration to humans.