Simple method for the routine determination of betaine and n,ndimethylglycine in blood and urine maurice d. Research open access simultaneous determination of. Betaine is a reagent that is used in soldering, resin curing fluxes, and organic synthesis. In this paper, we report the quantitative deter mination of click reaction with inhibition of. The recovery was satisfactory, ranging from 97 to 100%.
Determination of choline, betaine, and dimethylglycine in plasma by a highthroughput method based on normalphase chromatographytandem mass spectrometry. Determination of betaines by fast atom bombardment mass. The solution is now ready for the calorimetric procedure. Determination of protein the protein content of fresh plant materials was determined colorimetrically as described by lowry et al. Anhydrous betaine and 18crown6 were obtained from sigma chemical co. Glycine is an amino acid which contains both acidic and basic pk a s. It has been successfully applied to the analysis of 22 species of grasses. Tomato plants were treated by gb after 2 and 6 weeks from transplanting date at concentrations of 0, 5, 10 and 20 mml, as a foliar application. Here, we report a simple and sensitive isocratic hplcuv method for the determination of dmg and betaine in plasma and urine.
Trimethylglycine tmg is an amino acid derivative that occurs in plants. A new, quick and effective procedure for the isolation and determination of betaine from two. Pdf a reliable method for spectrophotometric determination of. Glycine betaine content of 22 species of cereals and grasses was determined by pyrolysisgas liquid chromatography assay. Determination of endogenous glycinebetaine and ectoine accumulation suspensions of p. Use of glycinebetaine and potassium ameliorate drought in wheat. The production of strawberries is important worldwide, where low temperature reduces that and change fruit quality. Glycine betaine and salicylic acid induced modification in productivity of two different cultivars of wheat grown under water stress. Paul received for publication, june 7, 1934 very few methods for the determination of glycine have been published.
For example, in marine organisms, ab levels are commonly in the range of 110. A procedure for determining glycine betaine levels in plants involving pyrolysisgas chromatography is described. Determination of glycine betaine contents the amount of glycine betaine was estimated by using the method designed by grieve and grattan 1983. The endogenous choline supply limits glycine betaine. The role of glycine betaine in the protection of plants. Glycine betaine is stable in vivo in plants and the ratelimiting step in its accumulation is its. Glycine betaine biosynthesized from glycine provides an. This dissociation will occur due to the titration done using the glycine solution.
Also the size of the spot can give quantitative information the bigger the spot, the bigger the. Bessieres ma, gibon y, lefeuvre jc, larher f 1999 a singlestep purification for glycine betaine determination in plant extracts by isocratic hplc. Highaffinity transporters are used to import osmolytes from extracellular sources and ressl et al. The method described is particularly suitable for a large number of samples. The samples were then filtered and the filtrates were diluted 1. Petronia carillo and yves gibon author affiliations petronia carillo department of life science, ii university. Extraction and analysis of osmolytes are essential steps for. It must be remarked that although betaines have never been determined in bee pollen, the presence of glycine betaine in tomato pollen was assessed by 1 hnuclear magnetic resonance in one not so recent study. Although it may be found in several plants and roots, such as the beta vulgaris family, present in typical diets, just a few analytical methods have been developed for its extraction from roots. Ionic compounds other than betaines were bound on a mixed bed of strong anion and weak cation exchange resins. Glycine betaine and carnitine were accumulated by t. Free amino acids and glycine betaine in leaf osmoregulation of.
The method entails derivatizing the quaternary ammonium compounds to increase their sensitivity to detection by fast atom bombardment mass spectrometry. Block rj 1955 a manual of paper chromatography and paper electropho resis. Carboxymethyl trimethylammonium hydrochloride cas number 590465. Seed treatment with glycine betaine enhances tolerance of. Petronia carillo and yves gibon author affiliations petronia carillo. It is now often called glycine betaine to distinguish it from other betaines that are widely distributed in microorganisms, plants, and animals. Effects of antifreeze proteins and glycine betaine on.
The journal of biological chemistry 8 1987 by the american society for biochemistry and molecular biology, inc. It is a small ntrimethylated amino acid, existing in zwitterionic form at neutral ph. To determine the ability of lo28 and lo28b to accumulate 14cglycine. Tomato plants were cultivated on 27 th of september and 1 st of october in the first and second seasons, respectively. Accumulation of the compatible solutes, glycinebetaine.
For k m determination, the glycine betaine concentration was varied from 0. Molecular basis of transport and regulation in the na. Because the blood samples were also used for the determination of total homocysteine, they were placed on ice after collection, and plasma was obtained without delay by centri. Betaine is one of most studied biologically active compounds, due its role in the main biological processes.
Glycine betaine and salicylic acid induced modification in. In this study, we used a physiological approach to investigate the mechanism by which glycine betaine is accumulated in osmotically stressed r. A variety of methods exist in the literature for the analysis of. Betaine, the trimethyl derivative of the amino acid glycine, is a metabolite of plant and animal tissues. The standard curve for glycinebetaine was made by placing. The aim of the paper was to determine nitrogen compounds contributing to leaf cell osmoregulation of spinach spinacia oleracea submitted to. Betaine has had only limited clinical use, but has not been linked to instances of serum enzyme elevations during therapy or to clinically apparent liver injury. Proline, glycine betaine, total phenolics and pigment contents in. Glycine betaine, carnitine, and choline enhance salinity.
Increasing the productivity of tomato plants grown in. Glycine betaine concentrations were determined in subsamples of shoot and root tissue using high performance liquid chromatography hplc by the method of carillo and gibon 2011. Capillary electrophoresis method for the simultaneous determination of tma and. Betaines as a class were separated from aqueous plant extracts by a three resin ion exchange procedure. Potential nutritional and physiological functions of. Betaine aldehyde is a substrate for choline dehydrogenase mitochondrial chemical structure. New approaches for extraction and determination of betaine.
The glycine betaine content of higher plants can be determined by rapid and simple isocratic highperformance liquid chromatography on a c18 column. The increase of glycine betaine balanced the decline in free amino acids, mainly replacing glycine and serine the precursors of glycine betaine in the osmotic adjustment of the cells. In many bacteria, plants, and animals, glycine betaine is synthesized from choline in two steps. Extraction and determination of glycine betaine initiating author name. Glycine betaine determination extract store at 20c standard.
Since then, many other betaines have been discovered, and the more specific name glycine betaine distinguishes this one. At research, it was aimed determination the effects of antifreeze protein type iii and glycine betaine on tolerance to low temperature 2. Glycine betaine and glycine betaine analogues in common foods. The plants tissues was finely ground, mechanically shaken with 20 ml of deionized water for 24 h at 25oc. The quaternary ammonium compounds, choline and betaine, and dimethylglycine dmg reside along a metabolic pathway linked to the synthesis of neurotransmitters and membrane phospholipids and to homocysteine remethylation and, therefore, folate status. Synthesis of the osmoprotectant glycine betaine in. It is based on an ionexchange chromatographic separation with a dowex 50x12 column and subsequent colorimetric determination of both betaines as reineckate. The amount of glycine betaine and trimethylamine noxide tmao was determined as described in valadezbustos et al. Pdf determination of betaines by fast atom bombardment. Opua, an osmotically regulated binding proteindependent transport system for the osmoprotectant glycine betaine in bacillus subtilis. This regulation establishes an exemption from the requirement of a tolerance for residues of glycine betaine cas reg.
Osmotic control of glycine betaine biosynthesis and. Determination of betaines by fast atom bombardment mass spectrometry. Laryea, folkert steinhagen, sandra pawliczek, and udo wendel a simple and convenient method using commercially available derivatization reagents is described for the. Starting from a complex dry plant, it consists of a single aqueous extraction followed by a purification over a sole cationic ionexchange resin ag1, oh before simple.
Exogenous application of glycinebetaine and potassium. Betaine or n,n,ntrimethylglycine was named after its discovery in sugar beet beta vulgaris in the 19th century. Determination of amino acids using thin layer chromatography. Lack of a convenient, highthroughput method for the determination of these compounds has prevented. The three most commonly examined include proline, mannitol and glycine betaine. Determination of glycine betaine by pyrolysisgas chromatography in cereals and grasses. It is extremely soluble in water but includes a non. By adding naoh to glycine, the proton ion will dissociate. Gb and ab occur at very different concentrations in biological samples, with gb being by far the dominant compound. A rapid, sensitive, and selective method for the determination of betaines is described and discussed. Intracellular accumulation of glycine betaine has been shown to confer an enhanced level of osmotic stress tolerance in rhizobium meliloti.
Nmethy lated glycine derivatives and effects on yields in low re actant concentrations. Purification and characterization of a glycine betaine binding protein from escherichia coli received for publication, april 14,1987 anne barron, jae ung jung, and merna villarejos. Patton from the division of agricultural biochemistry, university of minnesota, st. Quantitative determination of click reaction in the. Glycine betaine aldehyde, often simply called betaine aldehyde, is an intermediate in the metabolism of glycine, serine and threonine. The synthesis and accumulation of both the osmolytes in shoot always exceeded roots. Alanine betaine synthesis is not controlled by choline availability, because it is derived from. Purification and characterization of a glycine betaine. Simple method for the routine determination of betaine and.
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