Véronique Ferchaud-Roucher

Influence of the type of indigestible carbohydrate on plasma and urine short-chain fatty acid profiles in healthy human volunteers

Background/Objectives:Health effects of whole grain foods are becoming more evident. In this study, we analysed the short-chain fatty acid profiles in urine and serum derived from the colonic fermentation process of 13C-barley meals, prepared from barley grown under 13CO2 atmosphere.Subjects/Methods:In a crossover study, five volunteers ingested intact barley kernels (high content of non-starch polysaccharides (NSP) and resistant starch (RS)) and barley porridge (high content of NSP only). Using a newly developed stable isotope technology, we monitored 14 and 24 h postprandially 13C-acetate, 13C-propionate and 13C-butyrate in plasma and urine, respectively. The oro-cecal transit time (OCTT) of the meals was measured with the hydrogen breath test.Results:The OCTT was 6 h and did not differ between the two test meals. An increase of 13C-acetate was observed already early after ingestion of the meals (<6 h) and was attributed to early fermentation of the test meal. A rise in plasma 13C-propionate in the fermentation phase could only be detected after the porridge and not after the kernel meal. An increase in 13C-butyrate was only found in the fermentation phase and was higher after the barley kernels. Urine 13C-short-chain fatty acids data were consistent with these observations.Conclusions:The difference in the profiles of 13C-acetate, 13C-propionate and 13C-butyrate indicates that NSP combined with RS results in an altered fermentation profile than dietary fibre alone.

Oral citrulline does not affect whole body protein metabolism in healthy human volunteers: Results of a prospective, randomized, double-blind, cross-over study

BACKGROUND & AIMS Citrulline increases protein synthesis during refeeding in rodents with short bowel syndrome, aging and malnutrition, and improves nitrogen balance in fed healthy humans. The aim of the current study therefore was to determine whether citrulline had affected protein metabolism in healthy volunteers. METHODS In a randomized, double-blind, cross-over study, 12 healthy adults received a 5-h intravenous infusion of L-[1-(13)C]-leucine in the post-absorptive state, after a 7-day oral supplementation with 0.18 g/kg/day citrulline, or an iso-nitrogenous placebo. Treatment order was randomized, treatment periods were separated by 13-day wash out. Leucine appearance rate (Ra) was determined from plasma [1-(13)C]-keto-iso-caproate enrichment and leucine oxidation from expired (13)CO(2), and nitrogen balance was estimated from 6-h urinary urea excretion. RESULTS Compared with placebo, oral citrulline supplementation increased plasma citrulline, arginine and ornithine concentrations, but failed to affect albumin, transthyretin, free insulin and insulin-like growth factor (IGF)-1 plasma concentrations, urinary nitrate excretion, or nitrogen balance. Citrulline supplementation did not alter leucine Ra, leucine oxidation, nor whole-body protein synthesis. CONCLUSION In healthy, well nourished volunteers, oral citrulline increases plasma citrulline and arginine availability but does not affect whole-body protein kinetics in the post-absorptive state.

Cord Blood Glutathione Depletion in Preterm Infants: Correlation with Maternal Cysteine Depletion

Background Depletion of blood glutathione (GSH), a key antioxidant, is known to occur in preterm infants. Objective Our aim was to determine: 1) whether GSH depletion is present at the time of birth; and 2) whether it is associated with insufficient availability of cysteine (cys), the limiting GSH precursor, or a decreased capacity to synthesize GSH. Methodology Sixteen mothers delivering very low birth weight infants (VLBW), and 16 mothers delivering healthy, full term neonates were enrolled. Immediately after birth, erythrocytes from umbilical vein, umbilical artery, and maternal blood were obtained to assess GSH [GSH] and cysteine [cys] concentrations, and the GSH synthesis rate was determined from the incorporation of labeled cysteine into GSH in isolated erythrocytes ex vivo, measured using gas chromatography mass spectrometry. Principal Findings Compared with mothers delivering at full term, mothers delivering prematurely had markedly lower erythrocyte [GSH] and [cys] and these were significantly depressed in VLBW infants, compared with term neonates. A strong correlation was found between maternal and fetal GSH and cysteine levels. The capacity to synthesize GSH was as high in VLBW as in term infants. Conclusion The current data demonstrate that: 1) GSH depletion is present at the time of birth in VLBW infants; 2) As VLBW neonates possess a fully active capacity to synthesize glutathione, the depletion may arise from inadequate cysteine availability, potentially due to maternal depletion. Further studies would be needed to determine whether maternal-fetal cysteine transfer is decreased in preterm infants, and, if so, whether cysteine supplementation of mothers at risk of delivering prematurely would strengthen antioxidant defense in preterm neonates.

In situ NMR spectroelectrochemistry for the structure elucidation of unstable intermediate metabolites

AbstractIn situ NMR spectroelectrochemistry is presented in this study as a useful hybrid technique for the chemical structure elucidation of unstable intermediate species. An experimental setting was designed to follow the reaction in real time during the experimental electrochemical process. The analysis of 1H NMR spectra recorded in situ permitted us (1) to elucidate the reaction pathway of the electrochemical oxidation of phenacetin and (2) to reveal the quinone imine as a reactive intermediate species without using any trapping reaction. Phenacetin has been considered as hepatotoxic at high therapeutic amounts, which is why it was chosen as a model to prove the applicability of the analytical method. The use of 1D and 2D NMR experiments led to the elucidation of the major species produced from the oxidation process. We demonstrated that in situ NMR spectroelectrochemistry constitutes a fast way for monitoring unstable quinone imines and elucidating their chemical structures. FigureIn situ NMR spectroelectrochemistry for drug metabolism studies

Effects of Extended-Release Nicotinic Acid on Apolipoprotein (a) Kinetics in Hypertriglyceridemic Patients

Objective—To determine the mechanisms by which extended-release nicotinic acid reduces circulating lipoprotein (a) concentrations in hypertriglyceridemic patients. Approach and Results—Eight nondiabetic, obese male subjects (aged 48±12 years; body mass index, 31.2±1.8 kg/m2) with hypertriglyceridemia (triglycerides, 226±78 mg/dL) were enrolled in an 8 week, double blind, placebo-controlled cross-over study. At the end of each treatment phase, fasted subjects received a 10 µmol/L per kg bolus injection of [5,5,5-2H3]-L-Leucine immediately followed by constant infusion of [5,5,5-2H3]-L-Leucine (10 µmol L−1 kg−1 h−1) for 14 hours, and blood samples were collected. A liquid chromatography–tandem mass spectrometry method was used to study apolipoprotein (a) (Apo(a)) kinetics. The fractional catabolic rate of Apo(a) was calculated with a single compartmental model using the apolipoprotein B100 (ApoB100) containing very low density lipoprotein tracer enrichment as a precursor pool. Extended-release nicotinic acid decreased plasma triglycerides (−46%; P=0.023), raised high-density lipoprotein cholesterol (+20%; P=0.008), and decreased Apo(a) plasma concentrations (−20%; P=0.008). Extended-release nicotinic acid also decreased ApoB100 (22%; P=0.008) and proprotein convertase subtilisin/kexin type 9 (PCSK9, −29%; P=0.008) plasma concentrations. Apo(a) fractional catabolic rate and production rates were decreased by 37% (0.58±0.28 versus 0.36±0.19 pool/d; P=0.008) and 50% (1.4±0.8 versus 0.7±0.4 nmol/kg per day; P=0.008), respectively. Conclusions—Extended-release nicotinic acid treatment decreased Apo(a) plasma concentrations by 20%, production rates by 50%, and catabolism by 37%. ApoB100 and PCSK9 concentrations were also decreased by treatment, but no correlation was found with Apo(a) kinetic parameters.

Simultaneous detection of stable isotope-labeled and unlabeled l-tryptophan and of its main metabolites, l-kynurenine, serotonin and quinolinic acid, by gas chromatography/negative ion chemical ionization mass spectrometry

A method for the detection of unlabeled and (15)N2 -labeled L-tryptophan (L-Trp), L-kynurenine (L-Kyn), serotonin (5-HT) and quinolinic acid (QA) in human and rat plasma by GC/MS is described. Labeled and unlabeled versions of these four products were analyzed as their acyl substitution derivatives using pentafluoropropionic anhydride and 2,2,3,3,3-pentafluoro-1-propanol. Products were then separated by GC and analyzed by selected ion monitoring using negative ion chemical ionization mass spectrometry. L-[(13)C11, (15)N2]-Trp, methyl-serotonin and 3,5-pyridinedicarboxylic acid were used as internal standards for this method. The coefficients of variation for inter-assay repeatability were found to be approximately 5.2% for L-Trp and (15)N2-Trp, 17.1% for L-Kyn, 16.9% for 5-HT and 5.8% for QA (n = 2). We used this method to determine isotope enrichments in plasma L-Trp over the course of a continuous, intravenous infusion of L-[(15) N2 ]Trp in pregnant rat in the fasting state. Plasma (15)N2-Trp enrichment reached a plateau at 120 min. The free Trp appearance rate (Ra) into plasma was 49.5 ± 3.35 µmol/kg/h. The GC/MS method was applied to determine the enrichment of (15)N-labeled L-Trp, L-Kyn, 5-HT and QA concurrently with the concentration of non-labeled L-Trp, L-Kyn, 5-HT and QA in plasma. This method may help improve our understanding on L-Trp metabolism in vivo in animals and humans and potentially reveal the relative contribution of the four pathways of L-Trp metabolism.

Acebutolol and alprenolol metabolism predictions: comparative study of electrochemical and cytochrome P450-catalyzed reactions using liquid chromatography coupled to high-resolution mass spectrometry

AbstractA comparative study of the electrochemical conversion and the biotransformation performed by the cytochrome P450 (CYP450) obtained by rat liver microsomes has been achieved to elucidate the oxidation mechanism of both acebutolol and alprenolol. For this purpose, a wide range of reactions such as N-dealkylation, O-dealkoxylation, aromatic hydroxylation, benzyl hydroxylation, alkyl hydroxylation, and aromatic hydroxylation have been examined in this study, and their mechanisms have been compared. Most of the results of the electrochemical oxidation have been found to be in accordance with those obtained by incubating acebutolol and alprenolol in the presence of CYP450, i.e., N-dealkylation, benzyl hydroxylation, and O-dealkoxylation reactions catalyzed by liver microsomes were found to be predicted by the electrochemical oxidation. The difficulty for the electrochemical process to mimic both aromatic and alkyl hydroxylation reactions has also been discussed, and the hypothesis for the absence of aromatic hydroxylated and alkyl hydroxylated products, respectively, for alprenolol and acebutolol, under the anodic oxidation has been supported by theoretical calculation. The present study highlights the potential and limitation of coupling of electrochemistry–liquid chromatography–high-resolution mass spectrometry for the study of phase I and phase II reactions of acebutolol and alprenolol. FigureThe electrochemical conversion versus the biotransformation catalyzed by CYP450

The differential apoA-I enrichment of preβ1 and αHDL is detectable by gel filtration separation

The aim of the study was to assess the isolation of HDL by fast protein liquid chromatography (FPLC) to perform kinetics studies of apolipoprotein (apo)A-I-HDL labelled with a stable isotope. Comparison between FPLC and ultracentrifugation has been made. ApoA-I-HDL kinetics were studied by infusion of [5.5.5-2H3]leucine for 14 h in five subjects. Using FPLC, preβ1 HDL and αHDL (HDL2 and HDL3) were separated from 200 μl of plasma samples. Total HDL was isolated by sequential ultracentrifugation (HDL-UC). The tracer-to-tracee ratio was higher in preβ1 HDL than in total HDL-UC. The higher leucine enrichment found in total HDL-UC compared to αHDL suggested the existence of a mixture of apoA-I-HDL sub-classes. From this difference in enrichments, the turnover rate of total HDL-UC, usually assumed to be αHDL, was probably overestimated in previous studies. To our knowledge, this study is the first report which provides a convenient tool to distinguish enrichments of apoA-I in preβ1 HDL and αHDL from total HDL previously used for kinetic measurements. This original and new method should help to understand the kinetics of HDL in humans and the reverse cholesterol transport dynamics.

Voltammetry coupled to mass spectrometry in the presence of isotope 18O labeled water for the prediction of oxidative transformation pathways of activated aromatic ethers: Acebutolol

The coupling between an electrochemical cell (EC) and a mass spectrometer (MS) is a useful screening tool (EC-MS) to study the oxidative transformation pathways of various electroactive species. For that purpose, we showed that the EC-MS method, carried out in the presence and absence of isotope (18)O labeled water leads not only to a fast identification of oxidation products but also leads to a fast elucidation of the mechanism pathway reaction. We examined herein the case of the electrochemical hydrolysis of activated aromatic ether. Acebutolol (β-blockers) was selected herein as model of activated aromatic ether, and its electrochemical oxidation was examined in both the presence and absence of isotope (18)O labeled water. To elucidate electrochemical hydrolysis pathway reaction: O-dealkylation or O-dealkoxylation, our approach was used to prove its applicability. The electrochemical oxidation mechanism was then elucidated showing an O-dealkoxylation reaction. In addition, density functional theory (DFT) calculations fully support the experimental conclusions.

Plasma ceramide, a real-time predictive marker of pulmonary and hepatic metastases response to stereotactic body radiation therapy combined with irinotecan.

BACKGROUND AND PURPOSES Early biomarkers of tumour response are needed to discriminate between responders and non-responders to radiotherapy. We evaluated the ability of ceramide, a bioactive sphingolipid, to predict tumour sensitivity in patients treated by hypofractionated stereotactic body radiation therapy (SBRT) combined with irinotecan chemotherapy. MATERIALS AND METHODS Plasma levels of total ceramide and of its subspecies were measured before and during treatment in 35 patients with liver and lung oligometastases of colorectal cancer included in a phase II trial. Cer levels were quantified by LC-ESI-MS/MS and compared to tumour volume response evaluated one year later by CT-scan. RESULTS Pretreatment plasma ceramide levels were not indicative of tumour response. Nevertheless, the levels of total ceramide and of its 4 main subspecies were significantly higher at days 3 and 10 of treatment in objective responders than in non-responders. According to Kaplan-Meier curves, almost complete tumour control was achieved at 1year in patients with increased total ceramide levels whereas 50% of patients with decreased levels experienced an increase in tumour volume. CONCLUSIONS Total plasma ceramide is a promising biomarker of tumour response to SBRT combined with irinotecan that should enable to segregate patients with high risk of tumour escape.