Amino Acid 15N/14N Analysis at Natural Abundances: A New Tool for Soil Organic Matter Studies in Agricultural Systems

Abstract

The effects of landuse, fertilizer history and soil type on the quantity and isotopic quality of hydrolysable soil amino acids were examined in 3 grassland and 2 arable soils. Results showed, (i) that overall concentrations of individual amino acids were highest in the grassland soils, (ii) that ‰δ¹⁵N values of the individual amino acids differed considerably between the five soils, and (iii) that the combination of amino acid ‰δ¹⁵N values and concentrations could be used to distinguish between landuse, crop type and fertilizer history. This preliminary study indicates that the pathways of transformation of soil amino acid N are influenced by long term N inputs and that associated biological processes are reflected in differences in concentrations and ‰δ¹⁵N values of individual soil amino acids.     

Precise δ13C-determination in the Range of Natural Abundance on Amino Acids from Protein Hydrolysates by Gas Chromatography - Isotope Ratio Mass Spectrometry

Abstract

Routine on-line ¹³C-analysis by coupled gas chromatography - combustion – isotope ratio - mass spectrometry of individual amino acids out of mixtures in the natural abundance range demands their strictly standardized derivatization. For the N-acetyl-propylesters of amino acids from protein hydrolysates and blood serum a mean precision of 0.5% ° (1 SD) for derivatization, separation and measurement could be attained. From δ¹³C-values of derivatives δ¹³C-values of amino acids could be calculated by a carbon balance equation, implying an isotope effect on the C-1 of acetate of about 1.04 for nine amino acids. The global δ¹³C-value of casein calculated from the individual amino acids differed by less than 1.5%° from the directly determined value. The δ¹³C-values of amino acids from plant protein hydrolysates analyzed by the on-line method were in agreement with results obtained by classical procedures. The method permits the δ-value determination of up to nine amino acids from 200 μg of a mixture in less than one hour. By this means the detection of ¹³C-labeled amino acids in nmole amounts diluted by the 5000-fold amount of carrier becomes possible.     

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?     

Untersuchungen zur Resorption und Metabolisierung oral applizierter 15N-markierter Nukleinsäuren

Es wurden Untersuchungen zur Resorption und Metabolisierung ¹⁵N-markierter Hefe-RNS an Ratten durchgeführt. Die Bilanz der ¹⁵N-Ausscheidung in Urin und Kot ergab eine nahezu vollständige Resorption und eine äuβerst niedrige Inkorporation des Nukleinsäurestickstoffs. Da für die Bewertung des Proteinstoffwechsels neben der Gesamt-¹⁵ N-Ausscheidungsmenge im Urin die ¹⁵N-Menge der Harnstoff-bzw. Ammonium-Fraktion herangezogen wird, wurde die prozentuale Verteilung der Nukleinsäuremetabolite auf die Metabolitfraktionen im Urin untersucht. Aufgrund der dabei gewonnenen Erkenntnisse können die bei Proteinstoffwechseluntersuchungen unter Verwendung ¹⁵N-markierter Hefen bzw. nukleinsäurehaltigen Hefeproteinen erhaltenen Daten korrigiert werden.     

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.     

Preparation of Naturally 13C-Rich Free Fatty Acids (FFA) from Corn Oil and their Application in Metabolic Studies of Lactating Cows

Abstract

When the ¹³C/¹²C ratios in lactose and milk fat of normally fed healthy cows are compared to ¹³C/¹²C in feed a metabolic depletion of ¹³C in the milk constituents becomes obvious.

Due to a carbon isotope effect in the decarboxylation step of pyruvate to Acetyl-Co-A resulting in a ¹³C depleted acetate, depot fat and milk fat are especially reduced in ¹³C relative to ¹²C.

Under pathophysiological conditions after longer fasting periods or in ketosis, long chain fatty acids from depot fat are metabolized and their ¹³C depleted carbon atoms enter the citric acid cycle via Acetyl-Co-A. Then the intermediary products of this cycle become ¹³C depleted too and with a drain on the oxalacetate pool by gluconeogenesis ¹³C depleted carbon atoms are channeled into lactose.

This hypothesis is apt to explain a ¹³C/¹²C decrease in lactose of ketotic cows relative to healthy controls. It is supported by the observation that ¹³C-rich fatty acids from corn oil dosed orally to a ketotic cow increase the ¹³C/¹²C ratios in lactose. So a crossing-over of carbon atoms from long chain fatty acids into glucose and lactose seems highly probable.