Leucine Oxidation in vivo: Inter-and Intraindividual Variation in Healthy Subjects as Assessed by Oral L-[1-13C]Leucine Loads

Interindividual variability of leucine catabolism was studied in eight overnight fasted healthy volunteers by means of a controlled oral L-[1-¹³C]leucine loading test (5 mg/kg body weight). With the exception of total CO² expiration (CV = 5%),a considerable variation (CV generally < or ?10%)was observed with all other metabolic parameters: increase of leucine/2-oxoisocaproate in serum, ¹³C-enrichment in serum metabolites and ¹³CO² in expired air, and estimated oxidation rates. The degree of variation was only partially referable to individual differences. A comparable high degree of variability was found when two probands underwent multiple loading tests (n = 4). Possible reasons and implications are discussed.     

L-[1-13C]phenylalanine breath test in patients with chronic liver disease of different etiologies

The aim of this study was to compare the oxidation of l-[1-¹³C]phenylalanine (¹³C-PheOx) in patients with chronic liver failure due to different etiologies using l-[1-¹³C]phenylalanine breath test. Breath samples were collected before the administration of 100 mg l-[1-¹³C]phenylalanine, and every 10 min thereafter until completion of 1 h. Control subjects (n=9) presented a larger cumulative percentage of ¹³C dose recovery (CPDR) than patients (n=124) with chronic liver disease, regardless of the etiology (7.5±0.7 vs. 4.2±0.2, p=0.001). No differences in CPDR were found considering the Child-Pugh (CP) class or etiology: alcoholic (CP A=7.7±0.7, CP B=4.1±0.5, CP C=2.0±0.3), hepatitis C virus (CP A=5.4±0.5, CP B=4.0±0.2, CP C=2.2±0.3), hepatocellular carcinoma (CP A=5.5±1.6, CP B=3.6±1.8, CP C=2.2±1.0); or cryptogenic cirrhotic patients (CP A=7.4±1.5, CP B=4.4±0.4, CP C=2.1±0.7). Results confirm that ¹³C-PheOx decreases in patients with cirrhosis with respect to controls, notwithstanding the etiology.     

Zum Stand der Stoffwechselforschung mit 15N- und 13C-markierten Tracersubstanzen in der Kinderheilkunde (Protein Turnover Research Using 15N and 13C Labelled Substrates in Pediatrics)

Abstract

Measurements in protein turnover and in metabolism of amino acids and their degradation products by means of stable isotope labelled substrates have been increasingly applied in clinical research over the last years. In spite of numerous studies dealing with this topic, quite a few important insufficiently clarified methodical aspects remain. This refers, for instance, to the choice of suitable tracer substances, the difficulties in the determination of the excretion plateau and the validation of the oxidation rates as measured with individual-labelled amino acids with regard to the whole body protein synthesis. Such problems may become of decisive importance in special subjects, such as preterm infants and critically-ill patients.

Investigations into these issues conducted by our group have revealed that the protein turnover in the very small preterm infant is by no means as intensive as previously claimed. The utilisation of urea nitrogen for the whole body protein synthesis of the infant may assume substantial proportions under the conditions of marginal protein intake and of catchup-growth. Studies conducted by means of ¹⁵N-labelled bifidobacteria have pointed at the intensive substrate exchange existing between microflora and host.

Pediatric research has to be non-invasive. Consequently, methods based on arterio-venous differences in tracer concentrations and on muscle biopsies do not have very high priority in pediatric research. A search for references published in the last five years has shown, that ¹⁵N-glycine is still the most frequently used tracer substance. There is a tendency towards a further increase of cell culture experiments run with stable isotope labelled amino acids.

Clinical research groups increasingly turn their attention to stable isotopes and mass spectrometry. This impressively demonstrates the continuing importance of tracerkinetic methods in all branches of medicine.     

Effects of ergot alkaloids on liver function of piglets can be detected by the [13C]methacetin breath test irrespective of oral or intramuscular route of tracer administration

Ergot alkaloids (sum=total alkaloids, TA) originate from the phyto-pathogenic fungus Claviceps purpurea and might exert feed intake depressing and hepatotoxic effects on animals. The aim of the study was to evaluate TA effects on performance and liver function of piglets with the [¹³C]methacetin breath test and two routes of tracer administration (orally, p.o.; intramuscularly, i.m.). Two ergot batches were mixed into piglet diets resulting in 21 and 17 mg TA kg^?1 (Ergot-5 and -12, respectively) and compared with an ergot-free control diet. Feed intake was significantly depressed after feeding the ergot containing diets (p=<0.001). The time at maximum ¹³CO₂ exhalation (t _max) and the half-life (t _0.5) were not influenced by treatments and varied between 25 and 68 min after the p.o., and 28 and 62 min after the i.m. administration of [¹³C]methacetin, respectively. The cumulative ¹³C recovery (cPDR₃₀) was significantly lower due to feeding the diet Ergot-5 (6.6 %) compared with the Ergot-12 (8.8 %) and the control diet (9.7 %) irrespective of the route of tracer administration (p=0.044). As a discrimination of the diet effects through both tracer administration routes is possible, the i.m. application should be preferred in piglets as this causes less stress than the oral forced administration.     

Ultra trace determination of fluorobenzoic acids in reservoir and ground water using isotope dilution gas chromatography mass spectrometry

The accurate ultra-trace analysis of six fluorobenzoic acids (FBAs) via isotope dilution gas chromatography mass spectrometry through their deuterated analogues is described. North Sea reservoir and ground water samples were spiked with six deuterated FBAs (dFBAs), enriched using solid-phase extraction (SPE) and analysed using GC/MS after derivatisation with BF ₃· MeOH. All FBAs were enriched and determined simultaneously. SPE allowed a 250-fold enrichment of the acids if 100 mL of sample volume was used. The method enables the determination of FBAs down to the range of 8–37 ng L ^?1 with recoveries between 66 % and 85 %. It uses low amounts of chemicals and is adaptable to larger and smaller sample volumes.     

GC(Retention Indices), GC-MS,and 13C NMR of Two Citral-Rich Cymbopogon Leaf Oils: C. Flexuosus and C. tortilis

Leaf oils of Cymbopogon flexuosus and Cymbopogon tortilis were submitted to combined analysis by CG(retention indices), Gas Chromatography Mass Spectrometry (GC-MS), and ¹³C Nuclear Magnetic Resonance (NMR). The composition was dominated by geranial (39.2% and 32.0%) and neral (24.1% and 19.1%). Cis- and trans-7-hydroxy-3,7-dimethyl-3,6-oxyoctanal (synonym: cis- and trans-tetrahydro-5-(1-hydroxy-1-methylethyl)-2-methyl-2-furanacetaldehyde) were identified by ¹³C NMR by comparison with literature data.     

Evaluation of the oral 13C-bicarbonate tracer technique for the estimation of CO2 production and energy expenditure in dogs during rest and physical activity

For feeding of working dogs during their daily life, illness, routine jobs or sporting activities, an accurate determination of their nutritional requirements is essential to ensure their optimal health and performance. To predict the appropriate guidelines about how to feed dogs, it appears essential to determine the energy expenditure (EE) in a reliable and feasible way. In the present experiment, the non-invasive oral ¹³C-bicarbonate tracer technique (o¹³CT), i.e. collection of breath samples after oral administration of NaH¹³CO₃, was used for the estimation of CO₂ production and EE in dogs. Measurements were conducted during two days of rest, and during three days with 3 h of exercise per day. Average EE was 483 and 876 kJ kg^?0.75 d^?1 during rest and exercise, respectively. The o¹³CT seems appropriate to use as a minimal restrictive and non-invasive method to obtain reliable estimates of EE in dogs at different activity levels under near natural conditions.     

Liver function assessment with three 13C breath tests by two-point measurements

In this study, we performed three breath tests – l-[1-¹³C ]phenylalanine breath test (PBT), l-[1-¹³C ] methionine breath test, and [¹³C]methacetin breath test (MethaBT) – in patients with chronic liver disease to determine the optimal timing of expired air collection for diagnosing chronic liver disease and evaluating the grade of fibrosis. The subjects were 61 adults with normal livers, 98 chronic hepatitis patients, and 91 liver cirrhosis patients. We investigated the relationships of breath test results with routine biochemical tests and the Child–Pugh score, as well as the diagnostic capacities of the breath tests for liver dysfunction/cirrhosis and grade of liver fibrosis. For the diagnosis of liver cirrhosis and correlations with liver fibrosis, the accuracy of the PBT at 30 min (PBT30) was similar to that of the MethaBT at 15 min (Metha15). For liver function assessment by two-point measurement with ¹³C breath tests, we recommend the PBT30 and the Metha15.     

Development and application of 15N-tracer substances for measuring the whole-body protein turnover rates in the human,especially in neonates: a review

Our research group of the Children's Hospital of the University of Rostock (Rostock group) has long-time experience in ¹⁵N-labelling and in using yeast protein and its hydrolysates for tracer kinetic studies to evaluate parameters of the whole-body protein metabolism in premature infants. The particular advantage of applying an economically convenient, highly ¹⁵N-enriched, and completely labelled yeast protein for evaluating protein turnover rates is the fact that the ¹⁵N dose is spread among all proteinogenic amino acids. The absorption has been improved by hydrolysing [¹⁵N]yeast protein with thermitase into a mixture of amino acids, dipeptides and tripeptides so that faecal analysis becomes unnecessary when determining turnover rates. The review shows that, in contrast to the application of single ¹⁵N-labelled amino acids with resulting overestimation of protein turnover rates, the ¹⁵N-labelled yeast protein thermitase hydrolysate represents the amino acid metabolism more closely without causing amino acid imbalances. The ¹⁵N-labelled yeast protein thermitase hydrolysate leads to the estimation of reliable protein turnover rates, particularly in premature infants.     

Assessing the authenticity of commercial deep-sea drinking water by chemical and isotopic approaches

This study combines stable isotopes and chemical elements with statistical principal component analysis (PCA) to assess the authenticity of bottled commercial drinking water desalinized from deep seawater in the Taiwan market. Isotopic results indicate that true bottled deep-sea drinking water (DSDW) exhibits about 0?‰ for both δ²H and δ¹⁸O values, which are values similar to those of open seawater. By comparison, suspected counterfeit DSDW products display δ²H and δ¹⁸O values of around ?51?‰ and ?8?‰, respectively. These values are representative of terrestrial freshwater. In addition, suspected counterfeit DSDWs have δ and electrical conductivity values similar to a mixed water (MW) product that was manufactured by purifying terrestrial freshwater and adulterating this with small amounts of brine. Furthermore, PCA results indicate the chemical constitution of suspected DSDW products to be similar to the MW product which falls between purified terrestrial freshwater and desalinized open seawater. These similarities imply that suspected counterfeit DSDW products are manufactured in a similar manner to the declared MW product. This study demonstrates how combining knowledge of stable water isotopes and PCA can be used in assessing the authenticity of commercial DSDW products. The method should be of great interest to similar investigations elsewhere.     

Determination of Isotope Enrichment in 13C-or 2H-Labelled Branched-Chain l-Amino Acids From Physiological Fluids by Gas Chromatography-Mass Spectrometry: Use of l-Leucine Dehydrogenase for Specific Preparation of the Quinoxalinol Derivatives

A procedure is described which allows the specific and sensitive estimation of ¹³C- or ²H-label enrichment in branched-chain l-amino acids from physiological fluids: Amino acids are isolated from deproteinized samples by cation exchange chromatography. Specific conversion of branched-chain l-amino acids to their respective 2-oxo acids is achieved by treatment with l-leucine dehydrogenase from Bacillus sp. Reaction of the 2-oxo acids with o-phenylenediamin yields the branched-chain quinoxalinol derivatives which are purified by solid phase extraction. Isotopic label enrichment is then determined by gas chromatographic-mass spectrometric analysis of the O-trimethylsilyl quinoxalinol derivatives using an ammonia-chemical ionisation mode and selected ion monitoring of the quasi-molecular ions [MH]⁺ and [MH + 1]⁺. Applicability of the method is demonstrated by serum analysis in an oral loading test with l-[1-¹³C]leucine.     

Short-term carbon dynamics in a temperate grassland and heathland ecosystem exposed to 104 days of drought followed by irrigation

Temperate ecosystems are susceptible to drought events. The effect of a severe drought (104 days) followed by irrigation on the plant C uptake, its assimilation and input of C in soil were examined using a triple ¹³CO₂ pulse-chase labelling experiment in model grassland and heathland ecosystems. First ¹³CO₂ pulse at day 0 of the experiment revealed much higher ¹³C tracer uptake for shoots, roots and soil compared to the second pulse (day 44), where all plants showed significantly lower ¹³C tracer uptake. After the third ¹³CO₂ pulse (day 70), very low ¹³C uptake in shoots led to a negligible allocation of ¹³C into roots and soil. During irrigation after the severe drought, the ¹³C tracer that was allocated in plant tissues during the second and third pulse labelling was re-allocated in roots and soil, as soon as the irrigation started. This re-allocation was higher and longer lasting in heathland compared to grassland ecosystems.     

The effect of resistant starches on fat oxidation in healthy adults as measured by a 13CO2-breath test

A resistant starch (RS) mixture (MIX) consisting of fibre of potatoes (FP) and wrinkled pea starch (WPS), and high amylose maize starch (HAMS) were supplemented in adults to evaluate their effects on fat oxidation by means of a ¹³CO₂-breath test. Sixteen subjects received a regular diet either without or with the supplementation of MIX and HAMS in randomised order. After administration of a [U-¹³C]algal lipid mixture, exhaled air was collected over 14?h in 0.5- and 1-h intervals. The ¹³C abundances were measured by nondispersive infrared spectroscopy. In comparison to the dry run (DR), supplementation with MIX and with HAMS increased the cumulative percentage dose recovery: (DR: 16.7?%, MIX: 16.9?%, HAMS: 18.0?%), but without statistical significance. The colonic degradation of MIX and HAMS to short-chain fatty acids tends to lower the formation of carbohydrate-derived acetyl-CoA and contributes to a postprandial lipid oxidation increase by using fat-derived acetyl-CoA as a compensatory fuel source.     

Eu(III) complexation of phenyltrisalanine and phenylbisalanine in aqueous solution studied by photoluminescence and UV spectroscopy

The Eu(III)-coordination of phenyltrisalanine (Pta) 1, in aqueous solution was investigated. The photoluminescence data suggested that the chelating effect of Pta places the metal ion on the face of the aromatic ring as proposed in 4. Furthermore, Eu(III) is usually used as a substitute for Ca(II) in spectroscopic studies and the formation of a 1:1 complex between Pta and Eu(III) indicated the possibility that Pta may be used as a Ca(II) binder. For comparison, phenylbisalanine (Pba) 2, was subjected to similar studies and was found to behave more like phenylalanine.     

Aufnahme von 15NO2 und Einbau des 15NO2-Stickstoffs in verschiedene Stickstofffraktionen bei Sonnenblumen (Uptake of 15NO2 and Metabolic Transfer of the 15NO2 Nitrogen to Various Nitrogen Fractions of Sunflowers)

Abstract

Sunflowers were exposed to ¹⁵NO₂ at a range between 4,9 and 42,0 ppb. The ¹⁵NO₂ uptake was quantified and the fate of the ¹⁵N in different parts and pools of nitrogen was investigated. The very high δ¹⁵N-values of the free amino acid pool can't give an answer to the question whether the NO₂-nitrogen is incorporated by a different pathway, compared to the nitrogen which derives from root uptake. Or does a compartmentation of the nitrate pool in the plant cell cause a dilution of the ¹⁵N enrichment in the mineral nitrogen pool during sample preparation?     

Analysis of 13C-mixed Triacylglycerol in Stool by Bulk (Ea-IRMS) and Compound Specific (GC/MS) Methods

Abstract

This paper was presented in poster form at the 17th International Congress of Nutrition, August 27-31, Vienna. Austria (Annals of Nutrition & Metabolism 2001; 45(Suppl.1):349). Some of the data were also presented in poster form at the British Society of Gastroenterology Meeting, March 18-21, Glasgow, UK (Gut 2001; 48(Suppl.1):A91).

The ¹³C-mixed triacylglycerol (MTG) breath test is used to measure intraluminal fat digestion. In normal digestion. 20–40% of the ingested ¹³C label is recovered in breath CO₂. We aimed to identify the proportions of ingested label excreted in stool, as well as breath following ingestion of ¹³C-MTG by children with impaired exocrine pancreatic function and healthy controls. ¹³C enrichment of breath samples was measured by continuous flow isotope ratio mass spectrometry (IRMS) and cumulative percent dose recovered (cPDR) in 10 h was calculated. Total ¹³C of a faecal fat extract from each stool was measured by elemental analyser-IRMS, and ¹³C enrichment and concentration of the TBDMS derivative of octanoic acid was measured by GC/MS after hydrolysis of the fat extract. Stool 5-day cPDR was calculated. Mean breath cPDR was 35%. Mean cPDR in stool by combustion-IRMS and GC/ MS, respectively, was 0.8% and 1.0%. Therefore, the remaining 64% of the ¹³C label must remain in the body and variability in breath cPDR is due to postabsorptive rather than predigestive factors.     

The effect of physical back-diffusion of 13CO2 tracer on the coupling between photosynthesis and soil CO2 efflux in grassland

Pulse labelling experiments provide a common tool to study short-term processes in the plant–soil system and investigate below-ground carbon allocation as well as the coupling of soil CO₂ efflux to photosynthesis. During the first hours after pulse labelling, the measured isotopic signal of soil CO₂ efflux is a combination of both physical tracer diffusion into and out of the soil as well as biological tracer release via root and microbial respiration. Neglecting physical back-diffusion can lead to misinterpretation regarding time lags between photosynthesis and soil CO₂ efflux in grassland or any ecosystem type where the above-ground plant parts cannot be labelled in gas-tight chambers separated from the soil. We studied the effects of physical ¹³CO₂ tracer back-diffusion in pulse labelling experiments in grassland, focusing on the isotopic signature of soil CO₂ efflux. Having accounted for back-diffusion, the estimated time lag for first tracer appearance in soil CO₂ efflux changed from 0 to 1.81±0.56 h (mean±SD) and the time lag for maximum tracer appearance from 2.67±0.39 to 9.63±3.32 h (mean±SD). Thus, time lags were considerably longer when physical tracer diffusion was considered. Using these time lags after accounting for physical back-diffusion, high nocturnal soil CO₂ efflux rates could be related to daytime rates of gross primary productivity (R²=0.84). Moreover, pronounced diurnal patterns in the δ¹³C of soil CO₂ efflux were found during the decline of the tracer over 3 weeks. Possible mechanisms include diurnal changes in the relative contributions of autotrophic and heterotrophic soil respiration as well as their respective δ¹³C values. Thus, after accounting for physical back-diffusion, we were able to quantify biological time lags in the coupling of photosynthesis and soil CO₂ efflux in grassland at the diurnal time scale.     

The metabolic effect of resistant starch and yoghurt on the renal and faecal nitrogen and ammonia excretion in humans as measured by lactose-[15N2]ureide

Resistant starch (RS) and Lactobacillus acidophilus yoghurt (LC1) were supplemented simultaneously in healthy adults to evaluate the effect on the urinary and faecal nitrogen and ammonia excretion by means of lactose-[¹⁵N₂]ureide (¹⁵N-LU) degradation. Nineteen subjects received a regular daily diet either without or with supplementation of an RS-LC1-mixture composed of fibre of potatoes (RS type 1), wrinkle pea starch (RS type 2), and LC1 over a 20-day period in randomised order. Thereafter, ¹⁵N-LU was administered together with breakfast. Urine and faeces were collected over a period of 48 and 72 h, respectively. The ¹⁵N abundances were measured by isotope ratio mass spectrometry. The intake of the pre- and probiotic mixture composed of RS of type 1, type 2 and of LC1 significantly lowered the colonic generation and the renal excretion of toxic ¹⁵NH₃ and functioned as an ammonia shift from urinary to faecal ¹⁵N excretion when using ¹⁵N-LU as a xenobiotic marker.     

Synthesis,crystal structure and electrical properties of (C5H13NCl)2 SnCl6

In this work, a novel compound Bis(2-chloropropyl-N,N-dimethyl-1-ammonium) hexachloridostannate(IV) was synthesized and characterized by; single X-ray diffraction, Hirshfeld surface analysis, differential scanning calorimetric and dielectric measurement. The crystal structure refinement at room temperature reveled that this later belongs to the monoclinic compound with P2₁/n space group with the following unit cell parameters a = 7.2894(7) Å, b = 12.9351(12) Å, c = 12.2302(13) Å and β = 93.423 (6) °. The structure consists of isolated (SnCl₆)^2? octahedral anions connected together into layers via hydrogen bonds N–H….Cl between the chlorine atoms of the anions and the hydrogen atoms of the NH groups of the [C₅H₁₃NCl]⁺ cations. Hirschfeld surface analysis has been performed to gain insight into the behavior of these interactions. The differential scanning calorimetry spectrum discloses phase transitions at 367 and 376.7 K. The electrical properties of this compound have been measured in the temperature range 300–420 K and the frequency range 209 Hz–5 MHz. The Cole–Cole (Z′ versus Z″) plots are well fitted to an equivalent circuit model. The transition phase observed in the calorimetric study is confirmed by the change as function of temperature of electrical parameter such as the conductivity of grain (σ_g) and the σ_dc.