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A Lapidot

Publications and source records attributed to A Lapidot.

At least 55 records · Page 3Linked to original sources
PubMed1986

Metabolic pathways leading to liver glycogen repletion in vivo, studied by GC-MS and NMR.

A quantitative analysis of the pathways leading to glycogen repletion in rats was conducted. [U-13C]Glucose was administered intra-intestinally into awake fasted animals. The distribution of glucose isotopomers derived from liver glycogen, liver extracts and plasma was performed by GC-MS and 13C NMR. The potential gluconeogenic precursors for liver glycogen, lactate, alanine, glutamate and glutamine, were also analyzed. The amount of glycogen that is synthesized by the direct pathway was found to be 35%. The 13C enrichment of liver lactate, alanine and glucose is similar, indicating that they are the major precursors for liver glycogen synthesis via the indirect pathway. Our results demonstrate that after 24 h fasting, when glucose is supplied, gluconeogenesis from endogenous sources is not shut off.

Animals↗
PubMed1986

Dynamic aspects of amino acid metabolism in alloxan-induced diabetes and insulin-treated rabbits: in vivo studies with 15N and gas chromatography-mass spectrometry.

The present study was designed to determine the effect of alloxan-induced diabetes in rabbits on L-[15N]alanine and [15N]glycine kinetic parameters. This process was measured by single-dose administration of 15N-labeled amino acids to postabsorptive control rabbits and alloxan-induced diabetics and insulin-treated diabetic rabbits. Gas chromatography-mass spectrometry was used to determine the 15N enrichment of plasma glycine and alanine. Glycine and alanine pools and turnover rate constants were estimated from isotope enrichment time decay curves. The data from the present study indicate that plasma glycine and alanine turnover rate constants increased by 25-50% after alloxan administration but pool sizes showed only little changes, resulting in highly significant increases in fluxes and metabolic clearance rates of both alanine and glycine following alloxan administration; single-dose crystalline insulin or protamine zinc insulin treatment failed to restore the turnover rate constants of glycine or alanine toward control values and caused a depletion of 50% in glycine pool size; 7 days prolonged treatment with protamine zinc insulin restored alanine and glycine fluxes and metabolic clearance rates towards control postabsorptive values; and the reduction in flux values following insulin treatment is consistent with the reduction in the plasma glucose levels in rabbits. The data suggest that the regulatory mechanisms for uptake and metabolism of circulating glycogenic amino acids no longer are operative as a consequence of insulin deficiency following alloxan administration. Exogenous insulin restored the activity of the regulatory mechanism toward the postabsorptive control state.

Alanine↗
PubMed1985

Natural-abundance 13C nuclear magnetic resonance studies of regulation and overproduction of L-lysine by Brevibacterium flavum.

Natural-abundance 13C NMR spectroscopy has been used to study the metabolism of the L-lysine-producing bacterium, Brevibacterium flavum. Relationships of biomass formation, precursor uptake, and product excretion, as a function of culture medium, oxygen supply and specific cell membrane permeability, were rapidly determined using 67.89-MHz 13C NMR. The induction of lysine production throughout the growth cycle was studied. Intracellular and extracellular levels of free metabolites and unconsumed precursor were quantitatively measured as a function of growth culture conditions. Limited availability of oxygen resulted in accumulation and excretion of unfavorable products: lactate, succinate, alanine and valine. However, under optimal aeration conditions L-lysine was the sole metabolite detected extracellularly. Various important long-lived intermediates and storage compounds were detected in the intact cells (by NMR measurements). Carbon resonances of carbohydrates and amino acids were resolved and easily identified. Of particular interest are those of trehalose carbons, a storage carbohydrate. Natural-abundance 13C NMR spectroscopy seems most suitable for biotechnological processes where high concentrations of intermediates and end-products can be observed. We anticipate that this approach will be employed to screen overproducing bacterial strains.

Amino Acids↗
PubMed1985

Intramolecular interactions, mesomerism and dynamics in actinomycin D studied by 15N NMR spectroscopy.

We present a detailed conformational study of 15N-labelled actinomycin D in different organic solvents using 1H, 15N and two-dimensional (2D) NMR techniques at 30.4 MHz and 50.6 MHz. The assignment of the threonine and valine 15N resonances to the individual residues on the alpha- or beta-lactone rings was achieved via heteronuclear shift-correlated 2D NMR experiments. The solvent perturbation studies allow an estimation of the solvent accessibility of the nitrogens and carbonyl groups. Evidence is presented that the pentapeptide rings of actinomycin D have different conformations in polar and in apolar solvents. The chromophoric N10 is efficiently solvent-protected, the solvent-dependence of its 15N resonance resulting from solvent interactions at other positions of the molecule and from solvent-dependent changes in the twisting of the chromophoric system. The chromophoric 2-amino nitrogen is shown to exhibit a strong sp2 character due to the formation of a conjugated system with the carbonyl group at C1. Such a conjugation requires a non-planar chromophoric ring system. Additionally, a hydrogen bond connecting the 2-amino and the 1-carbonyl group was detected. In some solvents, two resonances appear for the 2-amino nitrogen implying the presence of the 2-amino group in two different conformations. The possible implications of the non-planarity of the chromophore for the intercalation process and for the biological activity of the drug are discussed.

Chemical Phenomena↗
PubMed1985

Microbial preparation of L-[15N]tyrosine and [15N]tyramine and their gas chromatographic-mass spectrometric analyses.

The preparation of L-[15N]tyrosine and [15N]tyramine by microbial synthesis is described. Immobilized Erwinia herbicola cells were added to a reaction mixture containing phenol, pyruvic acid, and 15NH4Cl. The reaction was driven by excess nonlabeled pyruvate and phenol. Under these denaturing concentrations of phenol, immobilized cells were more effective than free ones. Gram quantities of L-[15N]tyrosine were obtained without label dilution. The conversion of this L-[15N]tyrosine into [15N]tyramine by Streptococcus faecalis was performed at maximal efficiency. Gas chromatographic-mass spectrometric studies and 1H and 15N NMR analyses of the labeled compounds are reported.

Enterococcus faecalis↗
PubMed1984

Simultaneous determination of [2-15N]- and [5-15N]glutamine with gas chromatography-mass spectroscopy: applications to nitrogen metabolic studies.

A gas chromatographic-mass spectrometric method for analysis of L-[2-15N]- and L-[5-15N]glutamine is described. The method is based on direct acylation of glutamine with trifluoroacetic anhydride and the formation of the N,N-bis-trifluoroacetyl-L-glutamine derivative. This simple and sensitive method is capable of detecting approximately 0.5 atom% excess 15N in as little as 10 microliter of plasma with a mean coefficient of variance of 11.6%. The method was applied to determine the appearance of 15N enrichment in plasma amino-N and amide-N of glutamine in a healthy adult volunteer during a constant infusion of 15NH4Cl. A plateau level of 3.7 and 2.6 atom% excess was observed in amide-N and amino-N, respectively, at 1 and 2 h after 15NH4Cl infusion was started.

Acetic Anhydrides↗
PubMed1984

Actinomycin D, 1H NMR studies on intramolecular interactions and on the planarity of the chromophore.

The conformation of actinomycin D in acetone and chloroform solution at different temperatures has been studied by 1H NMR spectroscopy. At lower temperature the resonances due to the two chromophoric amino protons were observed. These signals exhibit very different resonance positions indicating a severely hindered rotation of the 2-amino group and the presence of a hydrogen-bond connecting the 2-amino and the 1-carbonyl groups. In 1H NMR spectra of partially 15N-enriched actinomycin D, the 1JN-H coupling constants at the 2-amino group were determined and a strong sp2 character for the 2-amino nitrogen was deduced. The strong amide character of the 2-amino group is caused by mesomerism involving the 1-carbonyl group. The amino proton signals are sensitive indicators for differences in the spatial relationship of the diverse parts of the actinomycin molecule. At lower temperatures a simultaneous and selective broadening of the alpha ring threonine and valine amide proton signals as well as of the 2-amino group resonance was observed, indicating the presence of one dynamic process in the molecule which slows down upon temperature reduction. A swinging motion of the N(10) nitrogen through the chromophore plane would explain this observation. The interpretation of these results requires the presence of a non-planar chromophoric system in the actinomycin molecule in acetone and chloroform solution. The possible implications of this non-planarity for the intercalation process and for the biological activity of the drug are discussed.

Amides↗
PubMed1984

The roles of insulin and glucagon in the regulation of amino acid turnover rate and pool size: in vivo study with [15N]glycine and gas chromatography--mass spectrometry.

Gas chromatography--mass spectrometry analysis of plasma amino acid derivatives has been used to determine the 15N enrichment time decay curves of plasma glycine following a single dose administration of [15N]glycine in untreated and insulins-, glucagon-, and cycloheximide-treated rabbits. The present study indicated the following: (a) Increases of 80 and 50% in plasma glycine disappearance rate constants occurred in insulin- and glucagon-treated rabbits as compared with control postabsorptive rabbits; (b) The hormones in the intact rabbits caused a significant depletion in glycine pool size, which led to a moderate reduction in the fluxes of glycine. (c) A significant reduction in glycine turnover rate constants and pool size was noted at 3 and 24 hr following the administration of a sublethal dose of cycloheximide and a restoration towards control postabsorptive values was observed 48 hr after cycloheximide administration. (d) Sublethal doses of cycloheximide inhibited by 60 and 90% the stimulatory action of insulin and glucagon on plasma glycine disappearance, respectively. The present data suggest that both insulin and glucagon may act directly on plasma glycine disappearance rates. The stimulatory action of insulin differs from the action of glucagon in that it is not completely blocked by cycloheximide. Presumably glucagon and insulin modify the glycine transport system at different sites or by a different mechanism.

Amino Acids↗
PubMed1984

Glycine pools and turnover rates in leukaemia patients measured with [15N]glycine.

Turnover parameters for plasma glycine were measured by administration of a single dose of [15N]glycine to overnight fasted healthy volunteers, nine patients with chronic leukaemia, one patient with acute monocytic leukaemia and one patient with chronic myeloid leukaemia before and after chemotherapy. Gas chromatography-mass spectrometry was used to determine plasma [15N]glycine enrichment. Pool sizes and turnover rate constants were estimated from time-decay curves for isotope enrichment. Turnover rate constants and metabolic clearance rates of plasma glycine in leukaemia patients were elevated in comparison with data for healthy volunteers. The increased rates of glycine disappearance from the circulation were not accompanied by a depletion in glycine pool size. Turnover rate constants fell during chemotherapy and were within the normal range during remission. There was a significant positive correlation between glycine turnover rate constants and leucocyte counts. The increase in glycine turnover rate constants may be related to proliferation of the neoplastic cells.

Female↗
PubMed1983

In vivo 15N NMR studies of regulation of nitrogen assimilation and amino acid production by Brevibacterium lactofermentum.

Glutamic acid producer Brevibacterium lactofermentum intact cells were used to demonstrate the feasibility of in vivo 15N NMR to follow nitrogen assimilation and amino acid production throughout the growth cycle. The induction of glutamic acid production by different growth conditions was studied. Intracellular and extracellular levels of free metabolites were estimated as function of oxygen supply and biotin concentration. 15N NMR enabled us to distinguish two phases during the fermentation. At the early stage of fermentation, glutamic acid was accumulated intracellularly independent of oxygen supply and no product was excreted. In the late growth phase, the permeability of the cells developed and L-glutamic acid was excreted. The effect of aeration and biotin concentration on cellular contents and excretion was also studied by 15N NMR. Glutamate, N-acetylglutamine, and glutamine were the main nitrogenous pools independent of cell culture conditions. Free ammonia was not accumulated intracellularly although glutamic acid fermentation can be characterized as the process of nitrogen assimilation and the uptake of ammonia is the key step. In conclusion, the application of in vivo 15N NMR spectroscopy unraveled various problems of nitrogen metabolism, in a rapid and nondestructive manner.

Amino Acids↗
PubMed1983

Microbial production of L-[15N]glutamic acid and its gas chromatography-mass spectrometry analysis.

L-[15N]Glutamic acid was prepared in high yields via a fermentative process. Brevibacterium lactofermentum, growing on a medium containing 97% enriched 15NH4Cl as a sole isotopic precursor, excreted mostly L-[15N]glutamic acid. The L-[15N]glutamic acid was purified and identified. Gas chromatography-mass spectrometry analysis was performed to demonstrate its usefulness in clinical studies.

Ammonium Chloride↗
PubMed1980

Regulation of pool sizes and turnover rates of amino acids in humans: 15N-glycine and 15N-alanine single-dose experiments using gas chromatography-mass spectrometry analysis.

Gas chromatography--mass spectrometry (GCMS) of plasma amino acid derivatives has been used to determine directly the 15N-enrichment of plasma glycine and alanine in ten volunteers at various metabolic states. Isotope-enrichment time-decay curves of plasma glycine and alanine, following a single intravenous dose of 15N-glycine or 15N-L alanine were obtained and provide an estimate of the extracellular compartment. Relatively narrow ranges were obtained for the glycine pool (7.7--11.8 micromole/100 g body wt), rate constants of transport (3.7--4.2 hr-1) and flux (28--43 micromole hr-1/100 g body wt) in the postabsorptive statcine pool (7.7--11.8 mumole/100 g body wt), rate constants of transport (3.7--4.2 hr-1) and flux (28--43 mumole hr-1/100 g body wt) in the postabsorptive state. In postprandial humans, pool sizes showed only a modest variation whereas the rate constants of transport of glycine and alanine were significantly lower. The plasma 15N-glycine and 15N-alanine isotope-enrichment time-decay curves over the first hour following a single i.v. dose of 15-amino acid represent mostly the hepatic uptake of glycine and alanine from the extracellular pool. The results presented in this study establish the stable isotope GCMS method as a more accurate, more convenient, and safe alternative to the use of radioactive labeled amino acids in studies of amino acid metabolism in human subjects.

Adult↗
PubMed1980

Glycine turnover rates and pool sizes in neonates as determined by gas chromatography-mass spectrometry and nitrogen 15.

This report makes use of a recent developed method with stable isotope and gas chromatography-mass spectrometry to determine the disappearance of labeled amino acids from plasma samples after iv administration of a single dose (93.3-mumoles/kg body weight) of [15N]glycine (92% N) in neonates. [15N]Glycine measurements were studied three times in each of six preterm infants at different gestational age and twice in two full-term infants. The first study was carried out in all infants 5 to 32 hr after delivery, the second study was performed on the third day of life, and the third study, included only the preterm infants, was at the age of 25 to 29 days. The isotope disappearance curves were linear within the first hr after [15N]glycine administration and represent mainly the hepatic uptake of glycine from the extracellular pool. The volume of glycine pool varied from the day of birth to 4 wk of life. Turnover rate constants of glycine ranging from 1.35 to 2.19 hr-1 were observed in preterm and term infants during the first 32 hr. Significant increases in turnover rate constants were noted on the third day of life in most infants. At 3 to 4 wk of life, statistically significant differences in pool size and turnover rate constants were obtained. An increase of 2- to 3-fold in turnover rate constants was observed as compared to day of birth. Pool sizes declined by 50%, but the resulted fluxes remained almost unchanged during the neonatal period. At the age of 3 to 4 wk, all these infants showed similar kinetic data as in adults.

Gas Chromatography-Mass Spectrometry↗