Enrichment and high-performance liquid chromatography analysis of tryptophan metabolites in plasma

Tryptophan metabolites with an indole ring are enriched by adsorption either as an ion pair with a trichloroacetic acid anion or as its undissociated form on porous polystyrene polymer (TSK 2000 S) from strongly acidic plasma deproteinized by trichloroacetic acid, and after washing with water, they are eluted with a 90% methanol solution. Following the removal of the solvent, the residue is dissolved in a small amount of water and then subjected to high-performance liquid chromatography (hplc) analysis. Using 0.2 ml of adsorbent, the recovery of the 500 pmol added for each of the tryptophan metabolites into 1.5 ml of deproteinized plasma is above 70%. This method is used for the analysis of normal rabbit and rat plasma. The hplc analysis, with native fluorescence detection, shows several peaks corresponding to tryptophan, 5-hydroxytryptophan, serotonin, 5-hydroxyindole-3-acetic acid, indole-3-acetic acid, and indole-3-propionic acid. Peak identification and cross reactivity were checked by the retention time with two hplc systems, fluorometric characterization, and electrochemical characterization. This method is easy and is simple enough for routine analysis.     

A tracking marker for the second dimension of the two dimensional gel electrophoresis of ribosomal proteins.

Conditions for the analysis of 7-methylguanine (7-MG) in the presence of major nucleic acid bases and other minor bases by means of high-performance liquid chromatography (hplc) were established. Purine bases were obtained by Chargaff's method to yield apurinic acid and were analyzed by hplc with strong cation-exchange resin, Zipax SCX. The method was applied to the determination of 7-MG in the liver DNA from rats treated with DMN and it was revealed that the results were comparable to those obtained by radioisotope method in terms of sensitivity and reproducibility. The lower limits of quantitation and detection of 7-MG were determined to be 10 and 4 ng, respectively.     

Changes in indole-3-acetic acid and abscisic acid levels during tomato (Lycopersicon esculentum Mill.) fruit development and ripening

Changes in the levels of indole-3-acetic acid (IAA) and abscisic acid (ABA) in tomato (Lycopersicon esculentum Mill.) fruit pericarp tissue during development through ripening were measured by GC-SIM-MS using d₃-ABA and ¹³C₆-IAA internal standards. In the two cultivars of fieldgrown tomatoes analyzed, the highest IAA levels (8–24 ng/g fw) were found at the earliest stage of development (7 days after anthesis) followed by a rapid decline in levels of the hormone. ABA levels of 40–60 ng/g fw were found at the earliest stages of development followed by a decline in levels until ripening occurred when elevated ABA levels (125 ng/g fw) were measured.     

High-pressure liquid chromatographic method for the determination of 25-hydroxycholecalciferol in cow plasma

A high-pressure liquid chromatographic (hplc) procedure was developed for the determination of 25-hydroxycholecalciferol (25-OH-D₃) in cow plasma or serum. The procedure involved extraction with an ethanol-ethyl ether mixture, separation of the aqueous phase, solvent partitions, column chromatography on silica gel, and, finally, determination by reversed phase hplc on a C₁₈-bonded microparticulate silica column. The identity of the drug in the extract was confirmed by comparison with a standard by liquid, thin-layer, and gas-liquid chromatography as the free steroid and the heptafluorobutyrate and by the uv spectra and also from the mass spectrum of the heptafluorobutyrate. Twenty-four samples from cows on normal diet (dry, lactating, and pregnant) were analyzed. The normal circulating levels of 25-OH-D₃ ranged from 40 to 58 ng/g; mean 48 ± 5.0 ng/g. The procedure was used to analyze a limited number of human and hog samples. Human serum contained 10–20 ng/g which was in agreement with literature values. Hog serum contained 18 ng/g.     

Quantitative determination of N-arylaceto- and N-arylglycolhydroxamic acids in biochemical reaction mixtures

A high-performance liquid chromatography (hplc) solvent system was developed for use with C₁₈ bonded packings that effected the chromatographic resolution of monoaromatic hydroxamic acids in complex mixtures. The success of this hplc system resulted from the incorporation of an aliphatic hydroxamic acid additive into the elution solvent. This additive, desferal mesylate, suppressed chemisorption effects between the stationary phase of the chromatographic packing and the aromatic hydroxamic acids being analyzed. The detectability limit for acetyl-derived aromatic hydroxamic acids was about 10 ng, while that for glycolyl-derived aromatic hydroxamic acids was about 5 ng. This level of sensitivity was sufficiently low to allow for the direct detection of these components in enzymatic reaction mixutres. By manipulation of the percentage methanol in the aqueous solvent the resolution of either type of hydroxamic acid from other related metabolites was readily accomplished.     

Dormancy in somatic embryos and seeds ofVitis: changes in endogenous abscisic acid during embryogeny and germination

Abscisic acid (ABA) in extracts of somatic embryos and seeds of Gloryvine (Vitis vinifera L.xV. rupestris Scheele) was measured by gas chromatography-mass spectrometry-selected ion monitoring using deuterated ABA, (±)-[C-3Me-²H₃]ABA, ([²H₃]ABA) as internal standard. The ABA content increased rapidly during embryogeny (0.035 ng/embryo at the globular stage to 0.22 ng/embryo at the mature stage). The level of ABA in the tissues of somatic embryos, expressed in ng/mg dry weight, decreased from the globular stage (0.76 ng/mg) to the mature stage (0.25 ng/mg). Chilling (4° C) induced normal germination of seeds and mature somatic embryos and precocious germination of globular, heart-shaped and torpedoshaped somatic embryos. In all cases chilling led to a marked reduction in endogenous ABA. Exogenous (±)-ABA inhibited the germination of chilled somatic embryos.Abbreviations ABA abscisic acid - [²H₃]ABA (±)-[C-3Me-²H₃]-abscisic acid - BHT 2,6-di-t-butyl-4-methylphenol - GC-MS gas chromatography-mass spectrometry - Me-ABA and Me-[²H₃]ABA methyl esters of ABA and [²H₃]ABA, respectively - SIM selected ion monitoring     

A rapid method for analysis of abscisic acid (ABA) in crude extracts of water stressed Arabidopsis thaliana plants by liquid chromatography--mass spectrometry in tandem mode.

We describe a quick, simple method for the extraction and quantification of the phytohormone (+)-abscisic acid (ABA) in samples of plants subjected to different water deficit treatments. The method includes an extraction with acetone/water/acetic acid (80:19:1, v/v), evaporation of the extracts and finally injection into the liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) system in multiple reaction monitoring (MRM) mode. The objective of this work has been to show the applicability of the method to quantify the endogenous content of ABA in Arabidopsis thaliana (Columbia, Col-0) leaves at three different degrees of water stress. Control plants, had almost constant low levels of ABA (2-3 ng g-1, f.w.) throughout the 3 weeks of the experiment. Nevertheless, stressed plants increase the ABA content between the first and the second week (from 10 to 21 ng g-1, f.w.). The results suggest that this method is useful for quantifying ABA from plant material and that it avoids tedious and time-consuming extraction, purification and/or derivatization processes.     

Retinoic acid: an endogenous compound of human blood. Unequivocal demonstration of endogenous retinoic acid in normal physiological conditions.

The occurrence of retinoic acid (vitamin A acid) as a normal constituent of the vitamin A reserve from the body is demonstrated. Improved methodologies based on selected peak monitoring (spm) and high-performance liquid chromatography (hplc) are used for the detection of retinoic acid in EDTA-plasma. After extensive cleanup by double-phase extraction and chromatography on Sephadex LH-20, retinoic acid is determined as its methyl derivative by spm. A different approach using double-phase extraction combined with a preextraction step and hplc is used to confirm the findings of the spm experiments. Both techniques proved the presence of retinoic acid in human plasma at a concentration of 1 to 3 ng/ml.     

A simple and efficient method for analysis of plant growth regulators: a new tool in the chest to combat recalcitrance in plant tissue culture

This report presents a simple, rapid and accessible validated method for quantification of eight major plant growth regulators (PGR): cytokinins (6-(γ,γ-dimethylallylamino)purine (2-iP), benzylaminopurine (BA) and zeatin), auxin (indole-3-acetic acid; IAA), jasmonic acid (JA), salicylic acid (SA), gibberellic acid (GA3) and abscisic acid (ABA) by liquid chromatography mass spectrometry. This method was tested in eight species including agricultural, ornamental and medicinal species: St. John’s wort, African violet, banana, American elm, tobacco, potato, sweet wormwood, and fennel. The method has good reproducibility and good sensitivity with %RSD (percent relative standard deviation) between 1 and 10% for all matrices and recovery values of 89 to 118% for all analytes. Method detection limits were 50.65 ng/g, 203.4 ng/g, 50.65, ng/g, 50.65 ng/g, 203.4 ng/g, 12.7 ng/g, 193 pg/g and 3.08 ng/g, for SA, IAA, zeatin, JA, GA3, ABA, 2-iP, and BA, respectively. Our results with a range of plant species show that this method represents a simple, low-cost method for analysis of PGRs, and may also serve as an useful starting point for the analysis of other related PGRs, as demonstrated by inclusion of the SA derivative, acetylsalicylic acid, and the JA derivatives: 12-oxo-phytodienoic acid and JA-isoleucine. The efficiency of this method will enable its incorporation into the plant tissue culture work flow and through characterization of endogenous PGR levels, will allow for improved method development for recalcitrant species facilitating fundamental and applied studies in plant morphogenesis, propagation and conservation.     

Wilt-induced ABA biosynthesis, gene expression and down-regulation of rbcS mRNA levels in Arabidopsis thaliana

The kinetics of wilt-induced abscisic acid (ABA) biosynthesis were investigated in shoots of Arabidopsis thaliana (L.) Heynh Landsberg erecta. ABA concentrations were measured using a radioimmunoassay (RIA) based on the monoclonal antibody MAC 252, and the RIA validated by comparison with combined gas chromatography-mass spectrometry using a [²H₃] labelled internal standard. The basal ABA content of Arabidopsis shoots was ca 10 ng g^?1 fresh weight; the concentrations had increased ca 4-fold within 30 min of the initiation of wilting, increased ca 8-fold after 4 h and 11-fold after 8 h. This stress-induced ABA production was dependent on de novo gene expression; pre-treatment of leaves and shoots with the metabolic inhibitors cordycepin and cycloheximide reduced the rate of subsequent stress-induced ABA biosynthesis from 12.5 ng g^?1 h^?1 to 1 ng g^?1 h^?1 and 0 ng g^?1 h^?1, respectively. In vitro translation of mRNA isolated from shoots subjected to wilting or ABA treatment followed by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed only minor changes. The effects of wilting and ABA on the content of total ribulose 1,5-bisphosphate carboxylase/oxygenase small sub-unit (rbcS) mRNA were also determined. Both wilting and exogenous ABA resulted in a substantial reduction in the amount of rbcS mRNA, an effect readily reversed by rehydration of wilted shoots. However, the effects of wilting were not mediated solely by newly-synthesised endogenous ABA, as wilting also reduced rbcS mRNA levels in the ABA-deficient aba-1 mutant, which did not produce ABA in response to loss of turgor. The amount of rbcS mRNA was higher in aba-1 shoots, suggesting that cellular rbcS mRNA levels are normally down-regulated by ABA. Cold treatment induced ABA production in wild type shoots only, but resulted in an increased rbcS mRNA content of both wild type and aba-1 shoots.     

Abscisic acid and apical dominance in Phaseolus coccineus L.

Abscisic acid (ABA) in lanolin, applied to the internode of decapitated runner bean plants enhances the outgrowth of lateral buds. The optimum concentration of the paste is 10^-5 M. The effect of ABA is counteracted by indoleacetic acid (IAA) but not by gibberellic acid (GA₃). There is no effect when ABA is applied to the apical bud or lateral buds of intact plants. However, 13.2 ng given to the lateral buds of decapitated plants stimulate their growth, whereas higher concentrations are inhibitory. Consequently, ABA enhances growth of lateral buds directly, but only when apical dominance is already weakened. The growth of the decapitated 2^nd internode was not affected by ABA. Radioactivity from [2-¹⁴C] ABA, applied to nonelongating 2^nd internode stumps of decapitated runner bean plants moves to the lateral buds, whereas [1-¹⁴C]IAA-and [³H]GA₁-translocation is much weaker. ABA transport is inhibited if IAA or [³H]GA₁ is applied simultaneously. In elongating internodes [¹⁴C]ABA is almost completely immobile. [¹⁴C]IAA-and [³H]GA₁-translocation is not affected by ABA. The amount of radioactivity from labelled ABA, translocated to the lateral buds, is highest during the early stages of bud outgrowth.Abbreviations ABA 2,4-cis, trans-(+)-abscisic acid - GA gibberellic acid - IAA indoleacetic acid - p.l. plain lanolin     

Stimulation by abscisic acid of the activity of phosphoenolpyruvate carboxylase in leaf disks of <Emphasis Type="Italic">Amaranthus hypochondriacus</Emphasis> L., C<Subscript>4</Subscript> plant: role of pH and protein levels

C₄ plants can more efficiently fix carbon in drought, high temperatures, and limitations of nitrogen or CO₂. Primary carboxylation is mediated by phosphoenolpyruvate carboxylase (PEPC, 4.1.1.31) in mesophyll cytosol of C₄ plants. Studies on hormonal regulation of C₄ PEPC have been quite limited. We have examined the activity/regulation of PEPC by abscisic acid (ABA), a plant hormone, in the leaves of Amaranthus hypochondriacus. PEPC activity was enhanced upon 1-h incubation with 20 μM ABA by about 30% in dark and more than 2-fold in light. Glucose-6-phosphate activation of PEPC was enhanced, and sensitivity to l-malate was decreased after ABA treatment. Butyric acid (a weak acid) decreased PEPC activity and restricted the stimulation by ABA. In contrast, methylamine (an alkalinizing agent) increased the PEPC activity and enhanced the effect of ABA. ABA increased the levels of PEPC protein as well as its mRNA. Butyric acid/methylamine modulated the changes induced by ABA of PEPC protein and mRNA levels, indicating that acidification/alkalinization of leaf disks was very important. Our results emphasize the marked modulation of PEPC in C₄ plants, by ABA. Such modulation by ABA could be significant when C₄ plants are under water stress, when ABA is known to accumulate. When present, cycloheximide decreased the PEPC protein levels and restricted the extent of activation by ABA. We conclude that the enhancement by ABA of PEPC activity depends on cellular alkalinization as well as elevated PEPC protein levels.     

Determination of endogenous and supplied deuterated abscisic acid in plant tissues by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry with multiple reaction monitoring

A specific, sensitive, and accurate method for determination of abscisic acid (ABA) in plant tissues is described. The method employs reversed-phase high-performance liquid chromatography and electrospray ionization-tandem mass spectrometry for multiple reaction monitoring of underivatized ABA and deuterated ABA analogs. Specific analogs were used to study the mechanism of ABA fragmentation, to select appropriate standards, and to identify compounds suitable for metabolic studies involving the supply of differentially labeled ABA. Limits of detection and quantification of 1.9 and 4.7 pg, respectively, were obtained over a linear calibration range of 0-1.5 ng ABA (on-column injected) using 5.8', 8', 8'-d(4) ABA as the internal standard. Accuracy and precision were within 15% for routine quality control samples. The method of standard additions, as applied to Arabidopsis thaliana seed extracts, was also used to validate the method for analysis of plant tissue samples. The utility of the method was further demonstrated by determining levels of ABA in western white pine seeds and of ABA and supplied 8', 8', 8', 9', 9', 9'-d(6) ABA in Brassica napus tissues, using 5.8', 8', 8'-d(4) ABA or 8', 8', 8'-d(3) ABA as the internal standard. Limits of quantification as low as 0.89 ng/g were achieved by optimizing the extraction procedure for each type of plant tissue.     

Abscisic Acid and Gibberellic Acid Contents in Ripening Barley Seeds

The ABA and GA3 contents were investigated in barley seeds during maturation and after harvest. The highest amount of ABA was found in milk and wax ripeness – 13 ng and 11 ng per seed respectively. The level decreased during the later stages of maturation and during release of dormancy and was 1 ng per seed 6 weeks after harvest. The content of gibberellic acid decreased in a similar way but in an earlier stage of maturation. The determined amounts of GA₃ were: 0.4, 0.1, 0.03 and about 0.05 ng per seed respectively, in milk, wax and full ripeness and after harvest. The results of quantitative determination, obtained with the GLC method, corresponded to the growth effects in the test of the first leaf of oat.     

Abscisic Acid Levels in Soybean Reproductive Structures during Development

Abscisic acid (ABA) concentrations and growth rates of developing soybean (Glycine max [L.] Merr. cv. Wye) seeds and pod walls were determined from anthesis to maturation using high pressure liquid chromatographic techniques. Developing soybean seeds contain up to 12,200 ng/g fresh weight of ABA compared to 330 ng/g fresh weight for pod walls. In the developing seeds ABA levels correlated with growth rates, being the highest during the most active growth period of seed enlargement, and then decreasing to less than 10 ng/g fresh weight at maturity. Higher levels of ABA were found to occur in the cotyledons and seed coats than the root-shoot axes at 21 days postanthesis. The time required for excised root-shoot axes to initiate growth in liquid culture decreased as seed development progressed and ABA levels of the seeds declined.     

Changes in levels of gibberellins,their putative conjugates,and ABA in zygotic embryos during late maturation and dry seed stages of <Emphasis Type="Italic">Brassica napus</Emphasis> cv Topaz

Two late stages [days 35 and 40 after pollination (DAP)] in zygotic embryo (ZE) development of Brassica napus were utilized to quantify, by the stable isotope-labeled dilution method, levels of “free” and “aglycone” gibberellins (GAs), as well as abscisic acid (ABA), during the programmed dehydration of the seed. GAs from both the early 13 hydroxylation and early non-hydroxylation pathways were present in these ZEs of B napus. Between 35 and 40 DAP endogenous ABA dropped precipitously (almost 30-fold) and this drop in ABA was accompanied by a significant reduction in levels of GA₁ and even in levels of the inactive GA catabolites, GA₈ and GA₂₉. Levels of GA₄ and putative GA₈₅ also dropped appreciably, though not significantly. In contrast, the levels of GA₂₀ and GA₉ (the immediate precursors of GA₁ and GA₄, respectively) did not change in the ZEs during this transition. A fungal-derived cellulase was used to hydrolyze the highly water-soluble fraction, which will contain GA conjugates. Relatively high levels of several GAs (GA₉, GA₂₀) were thus quantified after hydrolysis as the aglycones, e.g., 56 and 25 ng/g DW of GA₂₀ and 23 and 5 ng/g DW, of GA₉, respectively at DAP 35 and DAP 40. Other GAs found after hydrolysis of the highly water-soluble fraction remained relatively constant between 35 and 40 DAP. An exception was the putative GA₈₅ aglycone, which increased sixfold (free GA₈₅ decreased by ca. half). The transition to the dry seed stage for ZEs of B. napus is thus accompanied not only by the expected reduction in ABA, but also by reduced levels of many “free” GAs, especially the bioactive, 3β-hydroxylated GAs. In contrast, levels of 3-deoxy GAs remain relatively high, implying a partial block in the 3β-hydroxylation “activation” step of GA biosynthesis.     

Determination of 7-methylguanosine-containing 5'-termini of messenger ribonucleic acid by NaB[3H]4 labeling and high-performance liquid anion-exchange chromatography.

A method is described for the determination of 5′-terminal methylated (cap) structures in unlabeled mRNA based on oxidation with NaIO₄, reduction with NaB[³H]₄, cleavage with P₁ nuclease, and separation on a strong anion-exchange resin by high-performance liquid chromatography (hplc). Model compounds (cap 1 dinucleotides) were used to show that no structural alteration other than cleavage of the ribose ring of 7-methylguanosine occurred under the conditions used for oxidation and reduction. It was shown that the enzyme tobacco acid pyrophosphatase could be used to cleave cap dinucleotides containing unmodified or ring-opened ribose moieties and could also be used to release [³H]pm⁷G′ from NaB[³H]₄-labeled rabbit globin mRNA. All five known cap 1 dinucleotides were resolved by hplc. The cap of rabbit globin mRNA was identified as m⁷Gpppm⁶Am, in agreement with other methods of determination.     

A radioimmunoassay for abscisic acid

We have developed a radioimmunoassay (RIA) for abscisic acid (ABA) in the 0.1 ng to 2.5 ng range. Antibodies were obtained from rabbits immunized with ABA bound via its carboxyl group to bovine serum albumin. Cross-reactivity studies indicate that ABA esters are completely cross-reactive with ABA, while trans, trans abscisic acid (t-ABA) phaseic acid (PA) and dihydrophaseic acid (DPA) have much lower but significant cross-reactivities. Purification methods which reduce the levels of cross-reacting substances are described.Abbreviations RIA radioimmunoassay - DPA 4-dihydrophaseic acid - PA phaseic acid - GC gas chromatography - HPLC high performance liquid chromatography - TLC thin-layer chromatography - BSA bovine serum albumin - ABA abscisic acid - t-ABA trans, trans abscisic acid - IAA indoleacetic acid     

Osmotic stress, endogenous abscisic acid and the control of leaf morphology in Hippuris vulgaris L

Abstract. Previous reports indicate that heterophyllous aquatic plants can be induced to form aerial-type leaves on submerged shoots when they are grown in exogenous abscisic acid (ABA). This study reports on the relationship between osmotic stress (e.g. the situation encountered by a shoot tip when it grows above the water surface), endogenous ABA (as measured by gas chromatography-electron capture detector) and leaf morphology in the heterophyllous aquatic plant, Hippuris vulgaris. Free ABA could not be detected in submerged shoots of H. vulgaris but in aerial shoots ABA occurred at ca. 40ng (g fr wt)⁻¹. When submerged shoots were osmotically stressed ABA appeared at levels of 26 to 40ng (g fr wt)⁻¹. These and other data support two main conclusions: (1) Osmotically stressing a submerged shoot causes the appearance of delectable levels of ABA. (2) The rise of ABA in osmotically stressed submerged shoots in turn induces a change in leaf morphology from the submerged to the aerial form. This corroborates the hypothesis that, in the natural environment, ABA levels rise in response to the osmotic stress encountered when a submerged shoot grows up through the water/air interface and that the increased ABA leads to the production of aerial-type leaves.     

Validation of a radioimmunoassay for (+)-abscisic acid in extracts of apple and sweet-pepper tissue using high-pressure liquid chromatography and combined gas chromatography-mass spectrometry

A radioimmunoassay for (+)-abscisic acid (ABA) was developed and applied to the analysis of free ABA in extracts of apple (Malus pumila Mill.) and sweet pepper (Capsicum annuum L.) leaves at various stages during extract purification. Conjugates of ABA, were quantified after alkaline hydrolysis. The validity of the radioimmunoassay was tested by comparison of immunoassay estimates of ABA at different levels of extract purity with high-pressure liquid chromatography (HPLC) and combined gas chromatography-mass spectrometry. The antiserum, raised against (+)-ABA, was almost equally sensitive to (-)-ABA. Serum cross-reactivity with the methyl ester of ABA was 160% and with the glycosyl ester of ABA was 34%. Cross-reactivity with protein-ABA conjugates was very slight for C₄-conjugated keyholelimpet haemocyanin, but about 1000% for C₁-conjugated alkaline phosphatase. Other compounds tested showed extremely low or undetectable cross-reactivities. Further evidence for the specificity of the assay came from the agreement between the results using different assay methods for both apple and pepper extracts, and from the observation that the only zone of immunoreactivity on HPLC elution profiles corresponded with authentic (+)-ABA. The use of polyvinylpyrrolidone in the assay minimised interference by other substances in plant extracts. In pepper, free ABA levels increased rapidly during water stress and recovered to pre-stress levels within two days after rewatering. Levels of ABA conjugates were significantly lowr than free ABA in unstressed plants, and also increased rapidly with stress, although not to the same extent as free ABA, and did not recover as rapidly as did free ABA. In apple, levels of free ABA and of ABA conjugates both increased more than twofold over a two-week period of water stress. In contrast to pepper, however, immunoreactivity of the conjugate fraction was increased by hydrolysis, indicating that different ABA conjugates predominate in the two species.Abbreviations ABA abscisic acid - GC-MS combined gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography - Me-ABA methyl ester of ABA - PVP polyvinylpyrrolidone - RIA radioimmunoassay