Sample preparation techniques for the determination of natural 15N/14N variations in amino acids by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS)

In order to identify natural nitrogen isotope variations of biologically important amino acids four derivatization reactions (t-butylmethylsilylation, esterification with subsequent trifluoroacetylation, acetylation and pivaloylation) were tested with standard mixtures of 17 proteinogenic amino acids and plant (moss) samples using GC-C-IRMS. The possible fractionation of the nitrogen isotopes, caused for instance by the formation of multiple reaction products, was investigated. For biological samples, the esterification of the amino acids with subsequent trifluoroacetylation is recommended for nitrogen isotope ratio analysis. A sample preparation technique is described for the isotope ratio mass spectrometric analysis of amino acids from the non-protein (NPN) fraction of terrestrial moss. ¹⁴N/¹⁵N ratios from moss (Scleropodium spec.) samples from different anthropogenically polluted areas were studied with respect to ecotoxicologal bioindication.     

The Measurement of Muscle Protein Synthesis in Broilers with a Flooding Dose Technique: Use of 15N-Labelled Phenylalanine,GC-MS and GC-C-IRMS

Abstract

An experiment was carried out to measure fractional muscle protein synthesis rates (k _s ) in broilers with injection of a flooding dose of phenylalanine (1 ml/100 g body weight of 150 mM phenylalanine; 38 atom percent excess (APE) [¹⁵N]phenylalanine). K _s was calculated from the [¹⁵N] enrichment in phenylalanine of tissue-free and protein-bound phenylalanine using both gas chromatography mass spectrometry (GC-MS) and gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) for measurements after a 10 min isotope incorporation period.

The tertiary-butyldimethylsilyl (t-BDMS) derivatives of phenylalanine were used for gas chromatographic separation in both systems. GC-MS and GC-C-IRMS were calibrated for a range of 7 to 37 [¹⁵N]APE and 0 to 0.62 [¹⁵N]APE, respectively, and for sample sizes of 0.45 to 4.5 nmol phenylalanine and 7 to 40 nmol phenylalanine, respectively. Reproducibility of standards as a measure of precision varied from 0.06 to 0.29 [¹⁵N]APE and from 0.0004 to 0.0018 [¹⁵N]APE in GC-MS and GC-C-IRMS, respectively.

K _s was measured in the m. pectoralis major of broilers fed rye based diets (56%) which were provided either unsupplemented (-) or supplemented (+) with an enzyme preparation containing xylanase. K _s in breast muscles was significantly increased from 21.8%/d to 23.9%/d due to enzyme supplementation.

It can be concluded from the study that the measurement of protein synthesis in broilers with the flooding dose technique can be carried out by using [¹⁵N]phenylalanine, GC-MS and GC-C-IRMS.     

Incorporation of 15NO2 Nitrogen into Individual Amino Acids by Sunflowers Using Gc-C-Irms

Abstract

The nitrogen isotopic composition of individual amino acids in sunflower leaves after exposures to ¹⁵NO₂ in the range of ambient NO₂ concentrations (5–37 ppb) was analysed by Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS). Amino acids as well as the amides glutamine and asparagine were converted with MTBSTFA (N-methyl-N-(tert.-butyldimethylsilyl)-tri-fluoroacetamid) in pyridine to their corresponding TBDMS derivatives (N, O-tert.-butyldimethylsilyl) in a simple one-step silylation reaction. The derivatized amino acids were separated by gas chromatography, combusted on-line, and the products were sent continuously to an isotope ratio mass spectrometer. Accurate measurements were obtained, when more than 7 nmol N₂ were introduced into the ion source of the mass spectrometer per gas chromatographically separated and combusted compound. No interferences of the silicate and fluor containing derivatization agents on the performance of the system were observed.

In the range of ambient NO₂ concentrations sunflower leaves predominately incorporate the nitrogen derived from atmospheric NO₂ into soluble amino acids. The highest δ¹⁵N values were measured for alanine. The ¹⁵N enrichments of the detectable amino acids increased with increasing ¹⁵NO₂ concentration.     

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.     

Determination of 13C Enrichment by Conventional GC-MS and GC-(MS)-C-IRMS

Abstract

Isotopic enrichment of branched-chain L-amino acids (BCAA) and branched-chain 2-oxo acids (BCOA) in standard preparations and in human plasma samples withdrawn after oral loads with 1-¹³C labelled BCAA were measured on a conventional GC-MS system and an on-line GC-C-IRMS employing O-TMS quinoxalinol derivatives. It was concluded that the recently introduced GC-C-IRMS, owing to its high sensitivity, is the adequate analytical tool when tracer doses of stable isotope labelled compounds of low enrichment are to be used in biomedical in vivo studies.     

Sample preparation techniques for the determination of natural 15N/14N variations in amino acids by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS)

In order to identify natural nitrogen isotope variations of biologically important amino acids four derivatization reactions (t-butylmethylsilylation, esterification with subsequent trifluoroacetylation, acetylation and pivaloylation) were tested with standard mixtures of 17 proteinogenic amino acids and plant (moss) samples using GC-C-IRMS. The possible fractionation of the nitrogen isotopes, caused for instance by the formation of multiple reaction products, was investigated. For biological samples, the esterification of the amino acids with subsequent trifluoroacetylation is recommended for nitrogen isotope ratio analysis. A sample preparation technique is described for the isotope ratio mass spectrometric analysis of amino acids from the non-protein (NPN) fraction of terrestrial moss. 14N/15N ratios from moss (Scleropodium spec.) samples from different anthropogenically polluted areas were studied with respect to ecotoxicologal bioindication.     

The measurement of muscle protein synthesis in broilers with a flooding dose technique: use of 15N-labelled phenylalanine, GC-MS and GC-C-IRMS

An experiment was carried out to measure fractional muscle protein synthesis rates (k(s)) in broilers with injection of a flooding dose of phenylalanine (1 ml/100 g body weight of 150 mM phenylalanine; 38 atom percent excess (APE) [15N]phenylalanine). K(s) was calculated from the [15N] enrichment in phenylalanine of tissue-free and protein-bound phenylalanine using both gas chromatography mass spectrometry (GC-MS) and gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) for measurements after a 10 min isotope incorporation period. The tertiary-butyldimethylsilyl (t-BDMS) derivatives of phenylalanine were used for gas chromatographic separation in both systems. GC-MS and GC-C-IRMS were calibrated for a range of 7 to 37 [15N]APE and 0 to 0.62 [15N]APE, respectively, and for sample sizes of 0.45 to 4.5 nmol phenylalanine and 7 to 40 nmol phenylalanine, respectively. Reproducibility of standards as a measure of precision varied from 0.06 to 0.29 [15N]APE and from 0.0004 to 0.0018 [15N]APE in GC-MS and GC-C-IRMS, respectively. K(s) was measured in the m. pectoralis major of broilers fed rye based diets (56%) which were provided either unsupplemented (-) or supplemented (+) with an enzyme preparation containing xylanase. K(s) in breast muscles was significantly increased from 21.8%/d to 23.9%/d due to enzyme supplementation. It can be concluded from the study that the measurement of protein synthesis in broilers with the flooding dose technique can be carried out by using [15N]phenylalanine, GC-MS and GC-C-IRMS.     

The Influence of Ammonium on Nitrate Uptake and Assimilation in 2-Year-Old Ash and Oak Trees - A Tracer-Study with 15N

Abstract

Interactions between ammonium and nitrate as competitive N sources depend on various biotic and abiotic factors. The preference for one of these N sources and the influence of ammonium on nitrate uptake and nitrate reductase activity was investigated in a ¹⁵N labelling experiment using 2-year-old potted plants of ash (Fraxinus excelsior L.) and oak (Quercus robur L.) under greenhouse conditions.

Seedlings of both tree species use ammonium and nitrate in equal amounts when both N forms are supplied in a 1:1 ratio (1.5 mM NH₄ ⁺ + 1.5 mM NO₃ ^?), although there is a slight tendency that ammonium is preferred. In both species total N uptake is higher if ammonium and nitrate are supplied simultaneously when compared with uptake of nitrate alone (3 mM nitrate). If nitrate is the sole N source N uptake is only half as high as if ammonium and nitrate are supplied in a ratio of 1:1.

The distribution of nitrate reductase between shoot and roots is not influenced by the N-form: nitrate reductase activity is always highest in the roots of both species under the conditions of this experiment.

Xylem sap analyses showed that both species transport higher concentrations of amino acids than of nitrate from the roots to the shoot. The amino acid composition is independent of the type of N source. Furthermore, ash trees contain more nitrate in the xylem sap than oak trees, reflecting the higher N uptake and the higher nitrate reductase activity in the leaves of this species.     

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.     

15N Distribution in Wheat and Chemical Fractionation of Root-Borne 15N in the Soil

Abstract

Spring wheat plants were grown in split-root containers and labelled with ¹⁵N by fertilizing one compartment of the container with ¹⁵NH₄ ¹⁵NO₃ (95 at.-% ¹⁵N exc.). After the harvest, approx. 90% of the ¹⁵N incorporated by the plants were found in the shoots and 3% in the roots; approx. 7% had been released into the soil of the unlabelled compartment. The ¹⁵N in the soil of the unlabelled compartment was extracted with KCl and hydrolysed with HCl. Approx. 60% of the ¹⁵N was found in the hydrolysable organic N pool of the soil and 9% in the fraction of the soluble and exchangeable inorganic nitrogen.     

Application of 15N NMR Spectroscopy to Studies of the Intermolecular Interaction of Biomolecules

Abstract

Nitrogen nuclei are frequently located at the interaction sites of biomolecules; for example, amide nitrogens in the peptide are the key to maintaining the peptide backbone conformation by hydrogen bonding. Histidine, tryptophan, and arginine side chains contain nitrogen atoms which are often located at the active sites of enzymes. Heterocyclic compounds like purine and pyrimidine bases are substances essential to information transfer by base pair formation of nucleic acids. Also some other co-factors and dyes, such as flavins, porphyrins and chlorophylls, are nitrogen-containing substances which regulate energy tranduction in biological systems. Lecithin and phosphatidylethanolamine are the principal components of the phospholipids from biomembranes. To detect interaction sites and to study the interaction mechanism of these biomolecules, the use of nitrogen NMR seems promising. Although more than 99% of naturally occurring nitrogen element has the ¹⁴N nuclei, a disadvantage of the use of ¹⁴N magnetic resonance has been recognized. It has a spin quantum number I = 1 and the associated quadrupole moment provides a very broad resonance signal of about 100～1000 Hz. Thus, for detection of small changes of the chemical environment, use of ¹⁴N magnetic resonance is not adequate. The natural abundance of ¹⁵N nuclei with a spin quantum number I = 1/2 (which give a sharp resonance signal) is only 0.3% (Table 1). But recent developments in the instrumentation of NMR spectroscopy have made it possible to observe the resonance of the nuclei with low natural abundance. Fourier transform (FT) NMR can save thousands of times the accumulation time to improve the signal to noise ratio of ¹⁵N spectra [1-3]. Also superconducting magnets with wide bores have made possible the use of thick sample tubes of 25 mmΦ and observation of the ¹⁵N resonance of substances of low solubility [4]. In spite of such instrumental development, the observation of the 15N resonance is still not easy because of its low sensitivity; about of proton magnetic resonance. In the application of ¹⁵N NMR in biological systems, we often encounter quite low solubility of biomacromolecules and also sometimes need to measure the concentration dependency of ¹⁴N chemical shifts. For such experiments, enrichment of ¹⁵N nuclei in the molecules is required. Chemical syntheses starting from the simple ¹⁵N containing compounds as an ¹⁵N source and also biological syntheses by bacterial fermentation using the ¹⁵N source in culture media are employed for ¹⁵N enrichment. Enrichment at specific positions of biomolecules is useful for spectral assignments and also for analyses of the pathways of biosyntheses [5].     

Long term changes in the distribution and δ15N values of individual soil amino acids in the absence of plant and fertiliser inputs

The long-term ‘biodegradation’ on soil amino acids was examined in the control plots of ‘42 parcelles’ experiment, established in 1928 at INRA, Versailles (France). None of the plots is cultivated, but is kept free of weeds, and mixed to a depth of 25 cm twice yearly. Topsoil (0–10 cm depth) samples collected in 1929, 1963 and 1997 were subjected to acid hydrolysis (6 N HCl) for comparison. The distribution and δ¹⁵N natural abundance of 20 individual amino acids in the soils were determined, using ion chromatography (IC) and gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS). The total N and amino acid-N (AA-N), respectively, decreased by 54 % and 73 % in the period from 1929 to 1997. The average N loss was comparable for 1929–1963 (period 1) and 1963–1997 (period 2), but AA-N loss was three times faster in the former period. This significant reduction in total AA-N content was mirrored in the individual amino acids, which decreased by 74 %?±?1 % (ranging 58–89 %) between 1929 and 1997. The bulk δ¹⁵N values generally increased from 1929 to 1997, mainly associated with comparable or even higher increase of δ¹⁵N of the non-AA-N in the soil. The residence time (t _1/2, time in which half of N was lost from a specific soil pool) was ca. 65?±?5 years for the bulk soil, and comparable for periods 1 and 2. However, between periods 1 and 2 it decreased from 128 to 41 years in the non-AA pool, but increased from 59 to 92 years in the AA-N pool. Proline and amino acids that appear early in soil microbial metabolic pathways (e.g. glutamic acid, alanine, aspartic acid and valine) had relatively high δ¹⁵N values. Phenylalanine, threonine, glycine and leucine had relatively depleted δ¹⁵N values. The average δ¹⁵N value of the individual amino acids (IAAs) increased by 1δ unit from 1929 to 1997, associated with a similar rise from 1929 to 1963, and no change thereafter till 1997. However, the δ¹⁵N values of phenylalanine decreased by more than 7δ¹⁵N units between 1929 and 1997. The δ¹⁵N shift of IAAs from 1929 to 1963 and from 1929 to 1997 was not influenced by the relative amount of N remaining compared with the 1929 soil concentrations. The only exception was phenylalanine which showed decreasing δ¹⁵N associated with its decreasing concentration in the soil. We conclude therefore that in the absence of plant and fertiliser inputs, no change in the δ¹⁵N value of individual soil amino acids occurs, hence the original δ¹⁵N values are preserved and diagnostic information on past soil N (cycling) is retained. The exception was phenylalanine, its δ¹⁵N decreased with decreasing concentration from 1929 to 1997, hence it acted as a ‘potential’ marker for the land use changes (i.e. arable cropping to a fallow). The long term biological processing and reworking of residual amino acids resulted in a (partial) stabilisation in the soil, evidenced by reduced N loss and increased residence time of amino acid N during the period 1963–1997.     

The Effect of Catch Crops on the 15N Nitrogen Percolation and Leaching During the Early Winter Period

Abstract

Lysimeter experiments with application of ¹⁵N in growth chambers were used to investigate to what extent the growth of oil radish can prevent by temporary biological N conservation the nitrogen percolation and leaching during late autumn and early winter periods. It could be shown that the oil radish plants incorporated 47% of the applied ¹⁵N and thus reduced substantially the ¹⁵N percolation to the deeper soil layers (60–100 cm) and the ¹⁵N leaching losses. Before giving final recommendations, the fate of the ¹⁵N contained in the oil radish must be examined in the late winter and early spring periods, after freezing of the plants.     

Long term changes in the distribution and delta(15)N values of individual soil amino acids in the absence of plant and fertiliser inputs

The long-term 'biodegradation' on soil amino acids was examined in the control plots of '42 parcelles' experiment, established in 1928 at INRA, Versailles (France). None of the plots is cultivated, but is kept free of weeds, and mixed to a depth of 25 cm twice yearly. Topsoil (0-10 cm depth) samples collected in 1929, 1963 and 1997 were subjected to acid hydrolysis (6 N HCl) for comparison. The distribution and delta(15)N natural abundance of 20 individual amino acids in the soils were determined, using ion chromatography (IC) and gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). The total N and amino acid-N (AA-N), respectively, decreased by 54 % and 73 % in the period from 1929 to 1997. The average N loss was comparable for 1929-1963 (period 1) and 1963-1997 (period 2), but AA-N loss was three times faster in the former period. This significant reduction in total AA-N content was mirrored in the individual amino acids, which decreased by 74 % +/- 1 % (ranging 58-89 %) between 1929 and 1997. The bulk delta(15)N values generally increased from 1929 to 1997, mainly associated with comparable or even higher increase of delta(15)N of the non-AA-N in the soil. The residence time (t(1/2), time in which half of N was lost from a specific soil pool) was ca. 65 +/- 5 years for the bulk soil, and comparable for periods 1 and 2. However, between periods 1 and 2 it decreased from 128 to 41 years in the non-AA pool, but increased from 59 to 92 years in the AA-N pool. Proline and amino acids that appear early in soil microbial metabolic pathways (e.g. glutamic acid, alanine, aspartic acid and valine) had relatively high delta(15)N values. Phenylalanine, threonine, glycine and leucine had relatively depleted delta(15)N values. The average delta(15)N value of the individual amino acids (IAAs) increased by 1delta unit from 1929 to 1997, associated with a similar rise from 1929 to 1963, and no change thereafter till 1997. However, the delta(15)N values of phenylalanine decreased by more than 7delta(15)N units between 1929 and 1997. The delta(15)N shift of IAAs from 1929 to 1963 and from 1929 to 1997 was not influenced by the relative amount of N remaining compared with the 1929 soil concentrations. The only exception was phenylalanine which showed decreasing delta(15)N associated with its decreasing concentration in the soil. We conclude therefore that in the absence of plant and fertiliser inputs, no change in the delta(15)N value of individual soil amino acids occurs, hence the original delta(15)N values are preserved and diagnostic information on past soil N (cycling) is retained. The exception was phenylalanine, its delta(15)N decreased with decreasing concentration from 1929 to 1997, hence it acted as a 'potential' marker for the land use changes (i.e. arable cropping to a fallow). The long term biological processing and reworking of residual amino acids resulted in a (partial) stabilisation in the soil, evidenced by reduced N loss and increased residence time of amino acid N during the period 1963-1997.     

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.     

Relationships between Nitrogen Translocation and Accumulation in Growing Plant Parts of Grain Legumes

Abstract

Grain legumes absorbed mineral ¹⁵N at every stage of development and transported it directedly (just as symbiotically fixed ¹⁵N₂) into the plant parts growing at the respective stage. The ¹⁵N accumulation in the grains after long-lasting ¹⁵N supply can be ascribed, for the major part, to a secondary ¹⁵N translocation after a temporary incorporation into older plant parts (leaves, stem). Inhibition experiments with antibiotics revealed no direct relation between the accumulation of amino acid ¹⁵N in growing pods and seeds and the protein synthesis in this target organs. It may include, however, processes of (active ?) uptake and transport with a possible contribution of carrier systems specific for distinct amino acids.     

Investigation of Enterohepatic Bile Acid Circulation with [15N]Taurocholate

Abstract

[¹⁵N]Taurocholate has been prepared and used as a simple urine test substrate as a substitute for ¹⁴C- and ¹³C breath test substrates. [¹⁵N]Taurocholate was administered instead of [¹⁵N] glycocholate assuming that taurine is mainly used in the cholate cycle. Differences were found between healthy and diseased subjects, although deconjugated [¹⁵N]taurine was recovered in urine much less than expected. To get additional information on the actual excess of free taurine, [¹⁵N]taurine was administered orally in parallel investigations. Apparently, large fluctuations in the whole body taurine metabolism are responsible for the unexpected results.     

Deposition of 15N into Soil Layers of Different Proximity to Roots by Wheat Plants

Abstract

The translocation of root borne N compounds to different distances from the roots was studied by use of rectangular pots with three separated soil zones. Wheat plants were grown for 28 days (4 leaf stage) and subsequently pulse labelled by exposure to 15 ppm ¹⁵NH₃ (generated from (¹⁵NH₄)₂SO₄ with 95 at.-% ¹⁵N exc.) every other day with the rooting medium sealed from the atmosphere. Six pulses were applied in total.

The plants assimilated 65% of the label offered. The final ¹⁵N enrichment in the shoots was approx. 13 at.-% exc. and in the roots approx. 5 at.-% exc. These abundances were high enough to detect traces of ¹⁵N in soil approximately 1 cm distant from the roots. Most of the ¹⁵N recovered was retained in the shoots (about 90%), 5% were present in the roots and another 5% had been released into the rhizosphere. Considering the ¹⁵N released, 62% were found in the central root zone, 26% in the adjacent layer and 12% in the outer zone.     

Enzymatic synthesis of some 15N-labelled l-amino acids

Our group has developed a stereospecific enzymatic method, which is very efficient for the in vitro synthesis of l-[¹⁵N]serine, l-[¹⁵N]methionine and l-[¹⁵N]glutamic acid. These amino acids were prepared from the corresponding α -ketoacids in the suitable enzymatic systems. The bacterial NAD-dependent amino acid dehydrogenases alanin dehydrogenase, leucin dehydrogenase and glutamate dehydrogenase were used as catalysts. Glucose dehydrogenase was used for the regeneration of NADH and ¹⁵NH₄Cl as isotopically labelled material at 99 at.% ¹⁵N. All reactions are inexpensive and easy to perform on a synthetically useful scale (1-10g) giving high yields of l-amino acids. The ¹⁵N isotope content was determined by mass spectrometry.     

Estimation of Urea Production Rate With [15N2]Urea and [13C]Urea to Measure Catabolic Rates in Diabetes Mellitus

Abstract

For verifying catabolic states in insulin-dependent patients and dogs the method estimating urea production rates with ¹³C and with doubly ¹⁵N labeled urea, respectively, has been established. For a fast steady state of urea tracer dilution, a prime of 600 times the continuous infusion rate had to be injected. Urea was isolated from plasma samples by protein precipitation and cation exchange chromatography with a consecutive derivatization of the dried urea fraction (trimethylsilyl derivatives). The masses of the fragment ions m/z 189 (¹⁴N¹⁴N), 190 (¹⁴N¹⁵N) and 191 (¹⁵N¹⁵N) urea are monitored to estimate the [¹⁵N₂]urea frequency in the overall body urea pool in mol percent excess (MPE). 1 to 15 ng of derivatized urea were measured efficiently. An excellent correlation between expected standard and measured MPE (r = 0.9977) was achieved from solutions containing 1 to 7% [¹⁵N₂]urea. The interassay coefficient of variation amounted to < 10% for a [¹⁵N₂]urea portion of ≥ 3%.

Normoglycemic diabetic patients who were treated with insulin overnight showed significantly higher urea production compared to healthy controls (9.22 ± 2.07 vs. 5.4 ± 0.32 μmol·kg^?1 · min^?1; p < 0.05). Measurements in chronic diabetic dogs proved an increased rate of amino acid catabolism (+ 20% urea production) in systemic versus portal application of insulin in paired studies. This increased nitrogen load in diabetics may accelerate progression of diabetic nephropathy. - Thus, the established stable isotope technique may serve as a sensitive and useful indicator of amino acid catabolism in clinical and experimental research.