Sylvie Normand

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

SummaryThe 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. These results show that reliable kinetic parameters of glucose metabolism can be determined, without health hazard, in humans, at low cost, using 13C labelled glucose analysed with a new gas chromatograph-isotope ratio mass spectrometer.

The contribution of naturally labelled 13C fructose to glucose appearance in humans.

SummaryAmong monosaccharides, fructose has a small hyperglycaemic effect. In order to better explain the mechanisms which cause this metabolic property, we used tracers labelled with stable isotopes (deuterated glucose and naturally 13C labelled fructose) to quantify the overall glucose appearance, the rate of appearance in plasma of the 13C glucose synthesized from fructose, and the fructose oxidation in vivo in man during a 6-h period following ingestion of 0.5 and 1 g · kg−1 fructose. Fructose had a very small effect on overall glucose appearance (NS). During the 6 h of the study, it was found that the overall glucose appearance was 0.87±0.06 and 0.89±0.06 g · kg−1 (NS). The amount of glucose synthesized from fructose was 0.27±0.04 and 0.51±0.03 g · kg−1 (p<0.01) representing 31% and 57% of overall glucose appearance (p<0.01); the non-fructose glucose production was 0.60±0.02 and 0.38±0.03 g · kg−1 (p<0.05) after the 0.5 and 1 g · kg−1 load, respectively. Fructose oxidation was 0.28±0.03 and 0.59±0.07 g · kg−1 after the 0.5 and 1 g · kg−1 load respectively (p<0.01) representing 56% and 59% of the fructose loads (NS). These data show that the low hyperglycaemic effect of fructose is explained by its very small effect on overall glucose appearance and that fructose has a sparing effect on glucose metabolism.

On-line purification and carbon-13 isotopic analysis of carbon dioxide in breath : evaluation of on-line gas chromatography-isotope ratio mass spectrometry

Abstract An on-line gas chromatographic-isotope ratio mass spectrometric (IRMS) device consisting of a gas chromatograph, a column switching system, a continuous-flow dual inlet and an isotope ratio mass spectrometer is described. This device has been found suitable for the simultaneous on-line purification of expired breath CO 2 and the determination of its isotopic composition. The quality of the analytical data obtained validated the clinical use of the method to perform breath tests at low enrichment levels and from small samples. The whole procedure is reliable, simple and efficient. It is capable of analysing breath samples down to 50 μ1 within 150 s. Its high reliability and advantages over the conventional IRMS technique are discussed.

In vivo compartmental metabolism of13C docosahexaenoic acid, studied by gas chromatography-combustion isotope ratio mass spectrometry

The exchange of docosahexaenoic acid (22:6n-3) within lipid pools in rat and human has been followed as a function of time after the ingestion of triglycerides (TG) containing 22:6n-3 labeled with13C(13C 22:6n-3). The13C abundance in the fatty acid was measured by gas-chromatography-combustion isotope ratio mass spectrometry which allowed the detection of 0.001 atom13C percent12C. The13C 22:6n-3 appearance was rapid in the TG of very low density lipoprotein plus chylomicron fraction, in which the maximal labeling was observed at 3 and 2 h after ingestion in rat and human, respectively. Concomitant with the TG utilization of this fraction by lipoprotein lipase from tissues, unesterified13C 22:6n-3 appeared in the plasma albumin.13C 22:6n-3 bound to albumin was mostly present in unesterified form before 12 h post-ingestion while after that period, lysophosphatidylcholine (lysoPC) bound to albumin carried higher13C 22:6n-3 concentrations. These lyso-PC were mostly from hepatic origin and might represent a potential source of 22:6n-3 redistribution to tissues. The13C 22:6n-3 uptake into rat brain PC and phosphatidylethanolamine was still increasing when the concentration of plasma unesterified13C 22:6n-3 had already dropped to a minimal plateau value and during the period of maximal plasma circulation of13C 22:6n-3-lysoPC bound to albumin. In contrast, the uptake of13C 22:6n-3 into blood platelet PC occurred during the phase of important circulation of13C-22:6n-3 bound to albumin, suggesting thein vivo efficiency of the Lands pathway for this fatty acid. It is concluded that13C 22:6n-3 esterified in TG is rapidly absorbed and redistributed within plasma lipoproteins and that its redistribution within the two lipid species bound to albumin might influence its uptake by platelets and rat brain.

Effects of colonic fermentation on respiratory gas exchanges following a glucose load in man

Colonic fermentation produces short-chain fatty acids (SCFA). In humans, the amount of energy produced from the oxidation of these compounds is unknown and could modify the metabolic utilization of energetic fuels (eg, carbohydrates and lipids). If it were so, the equations used to evaluate the oxidation of nutrients from indirect calorimetry data should include the contribution of SCFA, which is not usually the case. Indeed, this fermentation process is usually considered as a minor and neglected energetic pathway. In this study, we have addressed the reliability of this assumption. Six normal subjects received orally either 50 g glucose or 50 g glucose plus 20 g lactulose. Their respiratory gas exchanges, breath hydrogen, methane, and 13CO2 concentrations, and plasma glucose, insulin, and free fatty acid (FFA) concentrations were monitored for 8 hours. CO2 production and breath hydrogen concentration were significantly greater with lactulose. No differences in oxygen consumption, breath 13CO2 production, or plasma concentrations of blood glucose, FFA, and insulin could be found between the two experiments. This suggests that the fermentation process induced by lactulose generates extra fuels going through an oxidation pathway. Therefore, the classic equations used to calculate carbohydrate and lipid oxidation and energy expenditure (EE) from indirect calorimetry data are probably not valid when fermentation is taking place. Indeed, in this experiment we could have overestimated glucose oxidation (12.5%) if the fermentation process were not considered. In conclusion, colonic fermentation in humans of nondigestible carbohydrates produces energetic substrates that could be used and oxidized as energetic fuels.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of glucose and amino acid infusion on glucose turnover in insulin-resistant obese and type II diabetic patients

Glucose turnover was assessed from [6,6-2H]glucose and [U-13C]glucose dilution analysis in six lean nondiabetic subjects, six obese patients with normal glucose tolerance, and six obese patients with non-insulin-dependent diabetes mellitus (NIDDM) during sequential infusions of glucose (13.9 mumol/kg fat-free mass [FFM]/min) and glucose+amino acid (4.2 mg/kg FFM/min). Cori cycle activity was assessed from the difference between glucose turnover obtained from [6,6-2H]glucose and [U-13C]glucose. During infusion of glucose alone, total glucose turnover was increased by 70% in obese NIDDM patients. Amino acid infusion decreased glucose concentrations by 0.8, 0.5, and 1.8 mmol/L in controls, obese patients, and NIDDM patients, respectively. This decrease in glycemia occurred despite an increase in glucose turnover in lean and obese nondiabetic subjects, and was due to an increased metabolic clearance rate (MCR) of glucose. In NIDDM patients the MCR of glucose was unchanged, and the decrease in glycemia was explained by a diminution in hepatic glucose output. Glucose turnover obtained by [6.6-2H] dilution analysis exceeded significantly the values obtained by dilution analysis in obese subjects and obese NIDDM patients, but not in controls. This indicates an increased Cori cycle activity in these patients.

Hypersensitivity to insulin during remissions in cyclosporin-treated IDDM patients

OBJECTIVE— To test the sensitivity to insulin in recent-onset IDDM patients, its course according to treatment, and the advent of remissions. RESEARCH DESIGN AND METHODS— The euglycemic hyperinsulinemic clamp was used in 54 recent-onset IDDM patients and 14 healthy control subjects. Patients were tested after 1,2, and 4 wk of treatment with either insulin or insulin plus cyclosporin A, during cyclosporin A-associated long-lasting remissions, and during relapses. RESULTS— Insulin sensitivity was markedly decreased in all patients at onset. It was rapidly restored by insulin therapy, whether immunosuppression was associated with it or not. Insulin sensitivity was even higher than normal in the remission patients, who also were characterized by the reappearance of some endogenous insulin secretion and the sustained normalization of blood glucose profiles. During relapses, the deterioration of the blood glucose profiles was associated with some loss of insulin sensitivity. CONCLUSIONS— Cyclosporin A-associated remissions represent an original situation that associates euglycemia with the persistence of low endogenous insulin secretion. Cyclosporin A by itself had no influence on sensitivity to insulin, but allowed the reappearance of some insulin secretory capacity that contributed, with the improvement of insulin sensitivity, to the development of the diabetes honeymoon. The secretion of endogenous insulin, although lower than normal, was sufficient to secure a high sensitivity to insulin and the maintenance of normal blood glucose profiles, presumably because of the fact that insulin was released directly into the portal vein in these conditions. This metabolic state was precarious: the optimal sensitivity to insulin disappeared in patients who relapsed. These results have important clinical consequences: the preservation of islet residual secretory capacity by the use of newer nontoxic immunosuppressive protocols, combined with a minimal supportive insulin therapy in remission patients, may prolong remissions and maintain an optimal insulin sensitivity.

Use of compounds naturally labeled with stable isotopes for the study of the metabolism of glycoprotein neutral sugars by gas-liquid chromatography-isotope-ratio mass spectrometry. Technical validation in the rat

In order to develop an alternative method to radioactive labeling for the study of the glycoprotein sugar metabolism in man, the possible use of stable isotopes provided by naturally, 13C-enriched dietary compounds has been explored in rat intestine and serum. Rats were fed a semisynthetic diet containing 67% wheat starch (containing 1.08692 13C atom/100 carbon atoms) for a week, and then the same diet containing corn starch (1.10042% 13C) for a week. Neutral sugars were prepared from delipidated, trichloroacetic acid-precipitable material from 200-400 mg of intestinal mucosa or 1 mL of serum, separated, and analyzed as alditol acetates by gas-liquid chromatography coupled to isotope-ratio mass spectrometry. This technique allowed the determination, in a single experiment, of the amount and 13C abundance of six neutral sugars (including xylose used as internal standard). Despite the low abundance of 13C in natural products, the sensitivity of the technique was found to be sufficient to detect isotopic enrichment as low as 0.001% with good accuracy and reproducibility in 2 micrograms of each glycoprotein neutral sugar. As an example, the pattern of labeling by dietary D-glucose from corn starch appears to be very different for fucose, ribose, mannose, galactose, and glucose of intestine or serum macromolecules.

Effects of specific dietary sugars on the incorporation of 13C label from dietary glucose into neutral sugars of rat intestine and serum glycoproteins.

Although theoretically all glycoprotein sugars can be derived from glucose, it may be hypothesized that specific dietary sugars could be preferential substrates for glycoprotein synthesis. To test this hypothesis, groups of rats received either continuously (continuous-labelling experiment) or for a single nutritional period (pulse-labelling experiment) a 13C-rich diet containing either maize starch or artificially labelled [13C]glucose. Some groups of rats were also provided during a single nutritional period with low amounts (20-200 mg/animal) of low-13C dietary sugars (mannose, galactose, fucose or fructose). If specific dietary sugars were preferentially incorporated into glycoproteins instead of glucose-derived labelled sugars, a decrease would be expected in the intestinal or serum glycoprotein-sugar 13C enrichment monitored by gas chromatography-isotope-ratio mass spectrometry (GC-IRMS). Contrary to this hypothesis the results showed no significant decrease with any of the specific dietary sugars. Furthermore, with dietary low-13C mannose or galactose, a significant increase in 13C enrichment of glycoprotein-sugars was observed compared with some other nutritional groups. Moreover, in the pulse-labelling experiment, dietary mannose and galactose induced similar patterns of 13C enrichment in intestinal and serum glycoprotein-sugars. Therefore, although specific dietary sugars do not appear to be preferential substrates for glycosylation under conditions and doses relevant to current concepts of nutrition, regulatory roles of some specific dietary sugars in relation to glycoprotein-sugar metabolism might be hypothesized. These findings could lead to similar studies using stable-isotope methodology in man which could have practical consequences, especially in parenteral nutrition where glucose is the only sugar provided to the metabolism.

Dietary specific sugars for serum protein enzymatic glycosylation in man.

All glycoprotein sugars can theoretically derive from glucose. However, dietary specific sugars could represent preferential substrates or have regulatory roles in enzymatic glycosylation. This hypothesis was tested in man using stable isotopes. Healthy subjects ingested different amounts (150, 300, or 550 mg) of artificially 13C-enriched sugar (galactose, mannose, or glucose) diluted in 200 mL water containing 50 g 13C-poor sucrose. 13C enrichment of expired CO2 was monitored for 8 hours during indirect calorimetry. Serum glycoproteins were precipitated and delipidated at various intervals. Glycoprotein neutral sugars were obtained by acidic hydrolysis, purified by ion-exchange chromatography, derivatized to alditol acetates, and analyzed by gas chromatography-isotope ratio mass spectrometry. The oxidation rate for galactose and mannose was slower than the rate for glucose. Total oxidation over the 8-hour period was less than 10% of the ingested amount of galactose or mannose. Galactose and mannose were readily incorporated into glycoprotein glycans, in the native form or after interconversion, despite ingestion of a large excess of sucrose: glycoprotein sugar 13C enrichment was strongly higher after 13C-galactose or 13C-mannose than after 13C-glucose. Thus, the metabolism of these three sugars appears to be different. Specific dietary sugars could represent a new class of non essential nutrients displaying interesting metabolic roles. This could have practical consequences especially in parenteral nutrition, where glucose is currently the only sugar available for metabolism.