Synchronized 13C-15N-NMR Spectroscopy to Follow Kinetics of Glycine Metabolization in the Rat Liver Non-Destructively

A method is proposed for reiterated measurements by turns of ¹³C and ¹⁵N NMR signals at intact rat liver tissue. Eight minutes after Wistar rats had been injected 1,2-¹³C; ¹⁵Nglycine, the liver was excised and stored in the triple resonance probe-head of a 2.1 Tesla NMR spectrometer. Alternatively ¹³C and ¹⁵N spectra were taken over some hours. Continued enzymatic activities of the liver tissue were seen in terms of metabolic changes of each of the carbon and nitrogen groups of glycine. Different half lives were found for the disappearance of the groups. The measurements gave insights into the kinetics and metabolism of an amino acid as an example to follow the vitality of an isolated organ non-destructively.     

Sulphur and Oxygen Isotopic Composition of Sulphates in Springs Feeding the Wieprz River and Other Springs of Lublin Upland and Roztocze

Abstract

Springs on Roztocze and Lublin Upland have been studied. Isotopic data are compared with data of chemical analyses. The results of studies allow us to distinguish five types of groundwaters. The differentiation is based upon different lithology; opokas, gaizes, sandy-silty-clay deposits, sands with shell sandstones, marly opokas, marly limestones and soft limestones of chalk type. A correlation can be observed between δ³⁴S and the concentration of Ca or Mg ions also a correlation between HCO₃ ^? ion concentration and δ¹⁸O in sulphates. Probably these correlations are the result of some simultaneous processes, which occur in groundwater. The seasonal variations of the isotopic composition and sulphate concentration were observed in four springs feeding the upper Wieprz. The variations were simultaneous and often similar in these springs. Probably, these variations are caused by the admixture of sulphates coming from shallow water layers (or leached from soil); however the variations of the groundwater level may also change chemical and isotopic composition in groundwater.     

Tracing the pathways of Neotropical migratory shorebirds using stable isotopes: A pilot study

We evaluated the potential use of stable isotopes to establish linkages between the wintering grounds and the breeding grounds of the Pectoral Sandpiper (Calidris melanotos), the White-rumped Sandpiper (Calidris fuscicollis), the Baird's Sandpiper (Calidris bairdii), and other Neotropical migratory shorebird species (e.g., Tringa spp.). These species molt their flight feathers on the wintering grounds and hence their flight feathers carry chemical signatures that are characteristic of their winter habitat. The objective of our pilot study was to assess the feasibility of identifying the winter origin of individual birds by: (1) collecting shorebird flight feathers from several widely separated Argentine sites and analyzing these for a suite of stable isotopes; and (2) analyzing the deuterium and ¹⁸O isotope data that were available from precipitation measurement stations in Argentina. Isotopic ratios (δ¹³C, δ¹⁵N and δ³⁴S) of flight feathers were significantly different among three widely separated sites in Argentina during January 2001. In terms of relative importance in separating the sites, δ³⁴S was most important, followed by δ¹⁵N, and then δ¹³C. In the complete discriminant analysis, the classification function correctly predicted group membership in 85% of the cases (jackknifed classification matrix). In a stepwise analysis δ¹³C was dropped from the solution, and site membership was correctly predicted in 92% of cases (jackknifed matrix). Analysis of precipitation data showed that both δD and δ¹⁸O were significantly related to both latitude and longitude on a countrywide scale (p < 0.001). Other variables, month, altitude, explained little additional variation in these isotope ratios. Several issues were identified that will likely constrain the degree of accuracy one can expect in predicting the geographic origin of birds from Argentina. There was unexplained variation in isotope ratios within and among the different wing feathers from individual birds. Such variation may indicate that birds are not faithful to a local site during their winter stay in Argentina. There was significant interannual variation in the δD and δ¹⁸O of precipitation. Hence, specific locations may not have a constant signature for some isotopes. Moreover, the fractionation that occurs in wetlands due to evaporation significantly skews local δD and δ¹⁸O values, which may undermine the strong large-scale gradients seen in the precipitation data. We are continuing the research with universities in Argentina with a focus on expanding the breadth of feather collection and attempting to resolve the identified issues.     

Estimation of protein synthesis rates using the flooding method and [15N] lysine

After injection of a single dose of double labelled lysine (1 MBq L-[u-¹⁴C]lysine and 150 μmol L-[α¹⁵N]lysine (95 atom% ¹⁵N)) to growing rats the fractional protein synthesis rates (FSR) of some organs were estimated by the “large dose-” or “flooding method”. Data obtained with both substances were compared and the following conclusions can be drawn:

—In principle lysine is suited as a flooding substance.

—In flooding experiments [¹⁴C]lysine and [¹⁵N]lysine gave identical FSR-values for the investigated organs.

—By application of [¹⁵N]lysine instead of the ¹⁴C labelled amino acid as flooding substance this method is suited for larger animals (pigs, sheep, ruminants) too, because of the absence of any radioactive burden.

Nach einmaliger Verabfolgung von doppelt markiertem Lysin (1 MBq L-[u-¹⁴C]Lysin und 150 μmol L-[α¹⁵N]Lysin (95 Atom% ¹⁵N)) an wachsende Ratten wurden nach der “Large dose-” oder “Flooding-Methode” die fraktionellen Proteinsyntheseraten (FSR) einiger Organe bestimmt und die für beide Substanzen erhaltenen Werte verglichen. Es ergaben sich folgende Schlußfolgergungen:

—Lysin ist grundsätzlich als Flooding-Substanz geeignet.

—[¹⁴C]Lysin und [¹⁵N]Lysin ergeben im Floodingversuch am gleichen Tier für die FSR der Organe identische Werte.

—Durch den Fortfall der radioaktiven Belastung bei Einsatz von [¹⁵N]Lysin anstelle von [¹⁴C]Lysin als Flooding-Substanz ist die Methode auch bei Croßtieren anwendbar.     

On the Metabolism of Dipterex

The metabolism of Dipterex in mammals, insects, plants, and microorganisms is discussed. Based on studies with P³²- and C¹⁴- labelled Dipterex the degradation of the insecticide and the different metabolic pathways are indicated. Whereas the detoxification of Dipterex in mammals and in cotton plant proceeds mainly via hydrolysis of the phosphonate C–P bond, its degradation in microorganisms invokes only O–CH₃ ester cleavage. In Prodenia larvae, Dipterex is metabolized preferentially (70%) by hydrolysis of O–methyl ester linkages and to a minor extent (30%) by splitting of C–P bond.     

Verschiebung des radioaktiven Gleichgewichtes 90Sr- 90Y im Organismus von Ratten nach oraler Verabfolgung

Bei unseren Stoffwechseluntersuchungen mit ⁹⁰Sr an Ratten und Kaninchen stellten wir wiederholt fest, daß sich die Aktivität einer Kot- oder Harnprobe verändert. Diese Veränderung der meßbaren Zählraten kann nur auf eine Verschiebung des radioaktiven Gleichgewichtes zwischen ⁹⁰Sr und ⁹⁰Y infolge der Stoffwechselvorgänge im Organismus zurückgefuhrt werden. Auf Grund der unterschiedlichen chemischen und physikalischen Eigenschaften von Strontium und Yttrium tritt bei jedem chemischen Eingriff eine Verschiebung des radioaktiven Gleichgewichtes zwischen ⁹⁰Sr und ⁹⁰Y ein. So bleibt beispielsweise bei der Fällung von Sr als Sulfat das ⁹⁰Y in Lösung und kann unter Einsatz von Trägermaterial fast quantitativ abgetrennt werden.     

The use of stable isotopes to evaluate water mixing and water use by flood plain trees along the Garonne valley

Before the confluence of the Tarn, the Garonne valley was the driest area in the entire south-west of France, due to the relatively low rainfall and low summer discharge of the Garonne River and its tributaries. The natural abundance of the stable isotope of oxygen (¹⁸O) and ionic charge of surface and ground water were used to estimate the water source for the Garonne River and phreatic subsurface water. We also measured these constituents in the sap of trees at several flood plain sites to better understand the source of water used by these trees. ¹⁸O signatures and conductivity in the Garonne River indicated that the predominance of water was from high altitude surface runoff from the Pyrenees Mountains. Tributary inputs had little effect on isotopic identity, but had a small effect on the conductivity. The isotopic signature and ionic conductivity of river water (δ¹⁸O: ?9.1‰ to ?9.0‰, conductivity: 217–410?µS/cm) was distinctly different from groundwater (δ¹⁸O: ?7.1‰ to ?6.6‰, conductivity: 600–900?µS/cm). Isotopic signatures from the sap of trees on the flood plain showed that the water source was shallow subsurface water (<30?cm), whereas trees further from the river relied on deeper ground water (>1?m). Trees at both locations maintained sap with ionic charges much greater (2.3–3.7×) than that of source water. The combined use of ¹⁸O signatures and ionic conductivity appears to be a potent tool to determine water sources on geographic scales, and source and use patterns by trees at the local forest scale. These analyses also show promise for better understanding of the effects of anthropogenic land-use and water-use changes on flood plain forest dynamics.     

Part 1: An Appraisal of New Radioisotope Techniques in the Study of Iodine Metabolism

The use of different radioiodines and technetium for the evaluation of the thyroid functions has been discussed. Preparation oe labelled iodotyrosines and labelled throxine is mentioned for the convey of different radiochemical tests and for the use of reference standards, necessary for the identification of separated amino acids. Paper chromatography and paper electrophoresis techniques are used for the identification and determination of iodinated amino acids or homones, either in the free state or bound to the proteins. Tc^99m is employed to substitute radioiodine in the study of uptake measurements of the thyroid gland and to evaluate the excretion of the isotope in urine. Thiourea-S³⁵ is employed, for the first time to human, to study the metabolism of the substance in different thyroid disorders.     

Astrocytic energy metabolism and glutamate formation--relevance for 13C-NMR spectroscopy and importance of cytosolic/mitochondrial trafficking

Glutamate plays a double role in ¹³C-nuclear magnetic resonance (NMR) spectroscopic determination of glucose metabolism in the brain. Bidirectional exchange between initially unlabeled glutamate and labeled α-ketoglutarate, formed from pyruvate via pyruvate dehydrogenase (PDH), indicates the rate of energy metabolism in the tricarboxylic acid (V_TCA) cycle in neurons (V_{PDH, n}) and, with additional computation, also in astrocytes (V_{PDH, g}), as confirmed using the astrocyte-specific substrate [¹³C]acetate. Formation of new molecules of glutamate during increased glutamatergic activity occurs only in astrocytes by combined pyruvate carboxylase (V_PC) and astrocytic PDH activity. V_{PDH, g} accounts for ∼15% of total pyruvate metabolism in the brain cortex, and V_PC accounts for another ∼10%. Since both PDH-generated and PC-generated pyruvates are needed for glutamate synthesis, ∼20/25 (80%) of astrocytic pyruvate metabolism proceed via glutamate formation. Net transmitter glutamate [γ-aminobutyric acid (GABA)] formation requires transfer of newly synthesized α-ketoglutarate to the astrocytic cytosol, α-ketoglutarate transamination to glutamate, amidation to glutamine, glutamine transfer to neurons, its hydrolysis to glutamate and glutamate release (or GABA formation). Glutamate-glutamine cycling, measured as glutamine synthesis rate (V_cycle), also transfers previously released glutamate/GABA to neurons after an initial astrocytic accumulation and measures predominantly glutamate signaling. An empirically established ∼1/1 ratio between glucose metabolism and V_cycle may reflect glucose utilization associated with oxidation/reduction processes during glutamate production, which together with associated transamination processes are balanced by subsequent glutamate oxidation after cessation of increased signaling activity. Astrocytic glutamate formation and subsequent oxidative metabolism provide large amounts of adenosine triphosphate used for accumulation from extracellular clefts of neuronally released K⁺ and glutamate and for cytosolic Ca²⁺ homeostasis.