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    Biological Processes

Medizin & Klinik

Die Durchführung von klinischen Studien, welche die biologischen Prozesse der menschlichen und tierischen Physiologie untersuchen, unterliegt der Frage nach der ethischen Vertretbarkeit. Die Stabilisotopenanalyse liefert einen nicht-invasiven Ansatz ohne Nebenwirkungen, die bei anderen Verfahren wie z.B. dem Einsatz von Radioisotopen auftreten können. Die Verwendung von angereicherten Isotopentracern mit künstlich hohen Häufigkeiten von kleineren Isotopen ist eine relativ einfache Methode, um subtile biologische Prozesse zu untersuchen.

Atemanalyse

Nachdem der Proband eine isotopisch markierte Mahlzeit (z. B. 13C markierte Glucose) eingenommen hat, zeigt das ausgeatmete CO2 des Patienten eine Anreicherung von 13C im Verhältnis zu 12C. Die Zeit, nach der die Anreicherung der 13C-Isotope im Atem auftritt, gibt einen Hinweis auf die Stoffwechselrate des Patienten. Bis zu 220 Atemproben können mit unserem iso FLOW Probenhandhabungssystem zur schnellen Analyse großer Probenzahlen analysiert werden.

Analyse von Körperflüssigkeiten

Die Verabreichung von doppelt markiertem Wasser (z.B. Wasser angereichert mit Deuterium und 18O) erlaubt es, den Gesamtenergieaufwand (Total Energy Expenditure, TEE) eines Subjektes (Mensch oder Tier) zu bestimmen. Die Hochleistungsisotopenanalyse von Deuterium und 18O von bis zu 180 Speichel-, Blut- und Urinproben kann mittels Headspace Equilibrierung mit dem iso FLOW Probenhandhabungssystem durchgeführt werden.

Biochemische Analysen

Um die komplexen physiologischen Prozesse innerhalb des Körpers besser zu verstehen, ist es notwendig, über die grobe Analyse von Atem und Körperflüssigkeiten hinauszugehen. Unsere Gas- und Flüssigchromatographie-Systeme (GC-IRMS und LC-IRMS) sind in der Lage, individuelle Stabilisotopenanalysen von Verbindungen durchzuführen, um mehr über diese komplexen Prozesse herauszufinden.

Medizinische Publikationen mit unseren Geräten

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37 Ergebnisse:

Preexercise galactose and glucose ingestion on fuel use during exercise
Medicine and Science in Sports and Exercise (2012)
John P. O'Hara, Sean Carroll, Carlton B. Cooke, Douglas J. Morrison, Thomas Preston, Roderick F G J King

Purpose: This study determined the effect of ingesting galactose and glucose 30 minutes prior to exercise on exogenous and endogenous fuel utilization during exercise. <p/>Methods: Nine trained male cyclists completed three bouts of cycling at 60% Wmax for 120 minutes, following an overnight fast. Thirty minutes prior to exercise the cyclists ingested either a fluid formulation containing placebo, 75g of galactose (Gal) or 75g of glucose (Glu) to which 13C tracers had been added, in a double blind randomized manner. Indirect calorimetry and isotope ratio mass spectrometry were used to calculate fat oxidation, total carbohydrate (CHO) oxidation, exogenous CHO oxidation, plasma glucose oxidation and endogenous liver and muscle CHO oxidation rates. <p/>Results: Peak exogenous CHO oxidation was significantly higher following Glu (0.68 +/- 0.08g[middle dot]min-1, P<0.05) compared to Gal (0.44 +/- 0.02 g[middle dot]min-1), however mean rates were not significantly different (0.40 +/- 0.03 vs. 0.36 +/- 0.02 g[middle dot]min-1, respectively). Glu produced significantly higher exogenous CHO oxidation rates during the initial hour of exercise (P<0.01), while glucose rates derived from Gal were significantly higher during the last hour (P<0.01). Plasma glucose and liver glucose oxidation at 60 minutes of exercise were significantly higher for Glu (1.07 +/- 0.1 g[middle dot]min-1, P<0.05 and 0.57 +/- 0.08 g[middle dot]min-1, P<0.01) compared with Gal (0.64 +/- 0.05 g[middle dot]min-1 and 0.29 +/- 0.03 g[middle dot]min-1, respectively). There were no significant differences in total CHO, whole body endogenous CHO, muscle glycogen or fat oxidation between conditions. <p/>Conclusion: The pre-exercise consumption of Glu provides a higher exogenous source of CHO during the initial stages of exercise, but Gal provides the predominant exogenous source of fuel during the latter stages of exercise and reduces the reliance on liver glucose.
Schlagworte: carbon , medi , liqfac

Urinary excretion of in vivo ¹³C-labelled milk oligosaccharides in breastfed infants.
The British journal of nutrition (2012)
Silvia Rudloff, Gottfried Pohlentz, Christian Borsch, Michael J Lentze, Clemens Kunz

Recent observations indicate that human milk oligosaccharides (HMO) are involved in a variety of physiological processes in infants. Their metabolic fate, however, is virtually unknown. We investigated metabolic aspects in infants after endogenous 13C-labelling of HMO. An oral bolus of natural and 13C-labelled galactose (Gal; 23 g Gal+4 g 13C-Gal) was given to ten lactating women. Aliquots of milk at each nursing as well as breath samples from the mothers and urine from their infants were collected over 36 h. The 13C-enrichment of HMO and their renal excretion was determined by isotope ratio-MS; characterisation was achieved by fast atom bombardment-MS. After the Gal bolus was given, an immediate 13C-enrichment in milk and in infants' urine was observed which lasted 36 h. Mass spectrometric analysis of 13C-enriched urinary fractions confirmed the excretion of a variety of neutral and acidic HMO without metabolic modification of their structures. Components with glucose split off at the reducing end were also detectable. Quantitative data regarding the infants' intake of lacto-N-tetraose and its monofucosylated derivative lacto-N-fucopentaose II ranged from 50 to 160 mg with each suckling, respectively; renal excretion of both components varied between 1 and 3 mg/d. Since the intake of individual HMO by the infants was in the range of several hundred mg per suckling, i.e. several g/d, and some of these components were excreted in mg amounts as intact HMO with the infants' urine, not only local but also systemic effects might be expected.
Schlagworte: medi , elem , gashead

Quantitation of plasma 13C-galactose and 13C-glucose during exercise by liquid chromatography/isotope ratio mass spectrometry.
Rapid communications in mass spectrometry : RCM (2011)
Douglas J Morrison, John P O'Hara, Roderick F G J King, Tom Preston

The utilisation of carbohydrate sources under exercise conditions is of considerable importance in performance sports. Incorporation of optimal profiles of macronutrients can improve endurance performance in athletes. However, gaining an understanding of the metabolic partitioning under sustained exercise can be problematical and isotope labelling approaches can help quantify substrate utilisation. The utilisation of oral galactose was investigated using (13)C-galactose and measurement of plasma galactose and glucose enrichment by liquid chromatography/isotope ratio mass spectrometry (LC/IRMS). As little as 100 μL plasma could readily be analysed with only minimal sample processing. Fucose was used as a chemical and isotopic internal standard for the quantitation of plasma galactose and glucose concentrations, and isotopic enrichment. The close elution of galactose and glucose required a correction routine to be implemented to allow the measurement, and correction, of plasma glucose δ(13)C, even in the presence of very highly enriched galactose. A Bland-Altman plot of glucose concentration measured by LC/IRMS against glucose measured by an enzymatic method showed good agreement between the methods. Data from seven trained cyclists, undergoing galactose supplementation before exercise, demonstrate that galactose is converted into glucose and is available for subsequent energy metabolism.
Schlagworte: carbon , medi , liqfac

Multi-element stable isotope analysis of H, C, N and S in hair and nails of contemporary human remains.
International journal of legal medicine (2011)
Christine Lehn, Elisabeth Mützel, Andreas Rossmann

This paper presents a comparison of the isotopic ratios of hydrogen, carbon, nitrogen and sulphur of three pairings of hair and nail tissue taken from contemporary human remains. Our aim was to examine the possibility of a direct comparison of isotopic data in hair with that of nail tissue for forensic purposes. The results indicate that stable isotope ratios of the elements were best comparable between human scalp hair longer than 3 cm and the distal end of the nails. There were no distinct variations between finger and toenails. Our isotopic data for bulk hair and nail confirmed that hair samples were slightly enriched in (13)C but depleted in (15)N compared to nail samples. Furthermore, our data reveal that δ(34)S values in nail samples were more variable than in hair samples. Direct comparison of the corresponding segments of hair and nail samples may be difficult due to individual differences especially for δ(15)N and δ(2)H. Hair may have an isotopic composition more consistent with the ingested food within a specific time than is provided by nail. It can be concluded that once a hair is formed, no further metabolic changes of the isotopic pattern should occur. Nevertheless, our data suggest that there was a change in isotope ratios particularly for δ(2)H along the hair shaft. Interpretation of the isotope data in corresponding segments of hair and nail for forensic purposes must consider particular variations, especially for chronological considerations.
Schlagworte: carbon , hydrogen , nitrogen , sulfur , medi , crim , elem

Effects of Static or Oscillating Dietary Crude Protein Levels on Fermentation Dynamics of Beef Cattle Diets Using a Dual-Flow Continuous Culture System
PLOS ONE (2011)
Paloma de Melo Amaral, Lays Débora Silva Mariz, Pedro Del Bianco Benedeti, Lorrayny Galoro da Silva, Eduardo Marostegan de Paula, Hugo Fernando Monteiro, Teshome Shenkoru, Stefanie Alvarenga Santos, Simon Roger Poulson, Antonio Pinheiro Faciola, K Doranal

The objective of this study was to evaluate the effects of increasing dietary crude protein (CP) levels and also comparing the effects of static versus oscillating dietary CP on ruminal nutrient digestibility, ruminal fermentation, nitrogen (N) metabolism, and microbial efficiency in beef cattle diets using a dual-flow continuous culture system. Eight fermenters (1,223 ± 21 mL) were used in a replicated 4 x 4 Latin square design with periods lasting 12 d each (8 d for adaptation and 4 d for sampling). Dietary treatments were: 1) 10% CP, 2) 12% CP, 3) 14% CP, and 4) 10 and 14% CP diets oscillating at 48-h intervals. Experimental diets consisted of 50% orchard hay and 50% concentrate. Fermenters were fed 72 g/d and solid and liquid dilution rates were adjusted to 5.5 and 11%/h, respectively. Data were analyzed using the MIXED procedure in SAS with α = 0.05. Apparent and true ruminal digestibilities of dry matter and organic matter were not affected (P > 0.05) by increasing dietary CP, nor by oscillating dietary CP. Total volatile fatty acids concentration and molar proportions of acetate, propionate, butyrate, valerate, iso-butyrate and iso-valerate were not affected (P > 0.05) by increasing or oscillating dietary CP. Ruminal NH3-N concentration increased linearly (P < 0.01) in response to increasing dietary CP. Total N, non-ammonia N, and rumen undegraded protein flows did not differ among treatments or between oscillating dietary CP and static 12% CP. Microbial N and NH3-N flows and microbial efficiency did not differ when comparing oscillating versus static CP (P > 0.05). However, there was a quadratic effect (P < 0.05) for these variables when dietary CP was increased. These results indicate that either ruminal microorganisms do not respond to oscillating CP levels or are capable of coping with 48-h periods of undernourishment.

Strong anion exchange liquid chromatographic separation of protein amino acids for natural 13C-abundance determination by isotope ratio mass spectrometry.
Rapid communications in mass spectrometry : RCM (2011)
Daniel a Abaye, Douglas J Morrison, Tom Preston

Amino acids are the building blocks of proteins and the analysis of their (13)C abundances is greatly simplified by the use of liquid chromatography (LC) systems coupled with isotope ratio mass spectrometry (IRMS) compared with gas chromatography (GC)-based methods. To date, various cation exchange chromatography columns have been employed for amino acid separation. Here, we report strong anion exchange chromatography (SAX) coupled to IRMS with a Liquiface interface for amino acid δ(13)C determination. Mixtures of underivatised amino acids (0.1-0.5 mM) and hydrolysates of representative proteins (prawns and bovine serum albumin) were resolved by LC/IRMS using a SAX column and inorganic eluents. Background inorganic carbon content was minimised through careful preparation of alkaline reagents and use of a pre-injector on-line carbonate removal device. SAX chromatography completely resolved 11 of the 16 expected protein amino acids following acid hydrolysis in underivatised form. Basic and neutral amino acids were resolved with 35 mM NaOH in isocratic mode. Elution of the aromatic and acidic amino acids required a higher hydroxide concentration (180 mM) and a counterion (NO 3-, 5-25 mM). The total run time was 70 min. The average δ(13)C precision of baseline-resolved peaks was 0.75‰ (range 0.04 to 1.06‰). SAX is a viable alternative to cation chromatography, especially where analysis of basic amino acids is important. The technology shows promise for (13)C amino acid analysis in ecology, archaeology, forensic science, nutrition and protein metabolism.
Schlagworte: carbon , medi , liqfac

Postprandial protein metabolism but not a fecal test reveals protein malabsorption in patients with pancreatic exocrine insufficiency.
Clinical Nutrition (2011)
Gheorghe Airinei, Claire Gaudichon, Cecile Bos, Cyriaque Bon, Nathalie Kapel, Bakhtiar Bejou, Jean Jacques Raynaud, Catherine Luengo, Thomas Aparicio, Philippe Levy, Daniel Tome, Robert Benamouzig

BACKGROUND & AIMS: Pancreatic exocrine insufficiency (PEI) impairs fat absorption, but few data are available on protein absorption. We investigated this question in patients with chronic pancreatitis, both in the absence and presence of enzyme therapy, using a stable isotope sensitive method. METHODS: Eleven patients with sustained PEI and regular enzyme substitution were investigated at hospital, after a washout period without enzyme substitution, and later after reintroduction of substitution. The digestibility and postprandial metabolism of dietary protein were characterized after the ingestion of a semi-synthetic single meal containing 20 g (15)N-labeled casein. RESULTS: At baseline, 20 ± 8% of dietary nitrogen was transferred to the metabolic pools vs. 24.5 ± 7% under enzyme treatment (P = 0.04). After treatment, the transfer of dietary nitrogen tended to increase in plasma amino acids, and increased significantly in plasma proteins and the deamination pool. In contrast, the fecal excretion of dietary nitrogen did not demonstrate any treatment effect. In patients not receiving insulin for diabetes, the treatment stimulated insulin secretion. CONCLUSIONS: Protein malabsorption was mostly undetectable using standard fecal tests. The study of the postprandial fate of dietary protein revealed a moderate increase of its transfer to metabolic pools after enzyme substitution.

Comparison of high-temperature conversion and equilibration methods for the determination of d31-palmitic acid oxidation in man using continuous-flow isotope ratio mass spectrometry.
Rapid communications in mass spectrometry : RCM (2011)
Valérie Sauvinet, Laure Gabert, Maud Alligier, Sylvie Normand, Hubert Roth, Martine Laville, Michel Désage

During nutritional interventions, the ingestion of d(31)-palmitic acid and H(2)(18)O allows the assessment of dietary fatty acid oxidation from cumulative (2)H recovery in urine and the estimation of the total body water pool (TBW) from (18)O dilution. Continuous-flow isotope ratio mass spectrometry (CF-IRMS) coupled to either equilibration or high-temperature conversion (HTC) techniques permits (2)H- and (18)O-enrichment measurements in biological fluids. Thus it was of great interest to compare these methods applied to the determination of dietary fatty acid oxidation. The linearity, accuracy and correlation between CF-equilibration and CF-HTC were first checked using (2)H- and (18)O-enriched water and urine samples. Urine samples from 14 subjects were then measured with both methods. The (2)H and (18)O raw data were normalised against calibration lines. The final aim was to study the impact of the normalised raw results on physiological data (i.e. TBW and d(31)-palmitate recovery). No significant difference was observed between the (18)O- and (2)H-enrichment measurements depending on the analytical method used. The TBW volumes calculated from the (18)O enrichments measured either with CF-equilibration or CF-HTC were not significantly different: respectively, 45.1 ± 1.0 L or 45.7 ± 1.0 L (mean ± sem, p = 0.09). The palmitic acid oxidation results obtained from the (2)H-enrichment measurements and the TBW from CF-equilibration vs. CF-HTC were not significantly different (p ≥ 0.26): with δ(2)H values of, respectively, 16.2 ± 1.6% vs. 16.2 ± 1.1% at 8 h, 18.7 ± 2.0% vs. 17.6 ± 1.3% at 12 h and 21.7 ± 1.9% vs. 21.5 ± 1.3% at 3 days post-dose (mean ± sem). Thus, even if CF-HTC was preferred because it was more practical to carry out, both methods allow the study of dietary lipid oxidation in man and generate similar results.
Schlagworte: hydrogen , oxygen , medi , gashead

Use of stable isotopes to measure the metabolic activity of the human intestinal microbiota.
Applied and environmental microbiology (2011)
Nicole Reichardt, Andrew R Barclay, Lawrence T Weaver, Douglas J Morrison

The human intestinal microbiota is a complex biological system comprising a vast repertoire of microbes with considerable metabolic activity relevant to both bacterial growth and host health. Greater strides have been made in the analysis of microbial diversity than in the measurement of functional activity, particularly in vivo. Stable isotope probing offers a new approach by coupling measurements of metabolic activity with microbial identification. Using a low-enrichment labeling strategy in vitro, this study has identified metabolically active bacterial groups via magnetic-bead capture methodology and stable isotope ratio analysis. Using five probes (EUB338, Bac303, Bif164, EREC482, and Clep866), changes in the activities of key intestinal microbial groups were successfully measured by exploiting tracers of de novo RNA synthesis. Perturbation of the nutrient source with oligofructose generated changes in the activity of bifidobacteria as expected, but also in the Bacteroides-Prevotella group, the Eubacterium rectale-Clostridium coccoides group, and the Clostridium leptum subgroup. Changes in activity were also observed in response to the medium type. This study suggests that changes in the functional activity of the gut microbiota can be assessed using tracers of de novo nucleic acid synthesis combined with measurement of low isotopic enrichment in 16S rRNA. Such tracers potentially limit substrate bias because they are universally available to bacteria. This low-enrichment labeling approach does not depend on the commercial availability of specific labeled substrates and can be easily translated to in vivo probing experiments of the functional activity of the microbiota in the human gut.

Strong anion-exchange liquid chromatography coupled with isotope ratio mass spectrometry using a Liquiface interface
Rapid Communications in Mass Spectrometry (2010)
Douglas J Morrison, Karen Taylor, Tom Preston

The introduction of liquid chromatography coupled with isotope ratio mass spectrometry (LC/IRMS) as an analytical tool for the measurement of isotope ratios in non-volatile analytes has somewhat simplified the analytical cycle from sample collection to analysis mainly due to the avoidance of the extensive sample processing and derivatisation that were necessary for gas chromatography/ combustion/isotope ratio mass spectrometry (GC/C/IRMS). Here we test the performance of coupling strong anion exchange to IRMS using only the second commercially available interface; the Liquiface. The system was modified from installation specification to improve peak resolution in the interface and maintain peak separation from the column to the mass spectrometer. The system performance was assessed by the determination of sensitivity, accuracy and precision attained from carbohydrate separations. The system performed satisfactorily after modifications, resulting in maintenance of peak resolution from column to mass spectrometer. The sensitivity achieved suggested that ?150 ng carbon could be analysed with acceptable precision (<0.3%). Accuracy was maintained in the interface as determined by correlation with offline techniques, resulting in regression coefficient of r2¼0.98 and a slope of 0.99. The average precision achieved for the separation of seven monosaccharides was 0.36%. The integration of a carbonate removal device limited the effect of background carbon perturbations in the mass spectrometer associated with eluent gradients, and the coupling of strong anion-exchange chromatography with IRMS was successfully achieved using the Liquiface
Schlagworte: carbon , medi , liqfac