Acidic Butanol Research Articles

Direct Quantitative Analysis of Lysophosphatidic Acid Molecular Species by Stable Isotope Dilution Electrospray Ionization Liquid Chromatography–Mass Spectrometry

In order to better understand the role of lysophosphatidic acid (LPA) in physiology and pathophysiology, it is necessary to accurately determine the molecular species and amounts of LPA in biological samples. We have developed a stable-isotope dilution, liquid chromatography–mass spectrometry assay for the direct quantitative analysis of 1-acyl-LPA. This method utilizes a deuterium-labeled internal standard, LPA (18:0-d35), and a single liquid–liquid extraction with acidic butanol that allows >95% recovery of LPA, followed by online normal-phase liquid chromatography–mass spectrometry. This protocol allows for the accurate, sensitive, and reproducible analysis of the individual 1-acyl-LPA species present in biological samples. The utility of the assay is demonstrated through the analysis of LPA species in plasma and serum from human volunteers. Total LPA in EDTA plasma was 0.61 ± 0.14 μM in males and 0.74 ± 0.17 μM in females, which increased to 0.91 ± 0.23 and 0.99 ± 0.38 μM after incubation for 24 h at 25°C. Total LPA in serum was 0.85 ± 0.22 μM in males and 1.57 ± 0.56 μM in females, which increased to 4.78 ± 0.89 and 5.57 ± 0.73 μM after incubation for 24 h at 25°C.

Low-molecular-weight proteins from sunflower (Helianthus annuus L.) seed: effect of acidic butanol treatment on the physicochemical properties

Sunflower seed proteins are not fully utilized as a food because they interact with chlorogenic acid (CGA) leading to development of an unfavourable colour. Low mol. wt. (LMW) proteins tend to be more closely associated with CGA. Washing with acidic butanol removes CGA from LMW proteins, but may alter their physicochemical properties. Effect of acidic butanol treatment (ABT) on LMW proteins was studied. LMW proteins were isolated from butanol treated and untreated sunflower seed flour and their properties compared. Sedimentation coeff. (1.8 S) was not affected by ABT. Gel filtration on Sephadex G-75 revealed 4 protein peaks. ABT increased the proportion of the 2nd peak from 35 to 53%. Mol. wt. of the proteins was not affected by ABT, as demonstrated by SDS-PAGE. Secondary structures of the native and ABT samples were (%): 19 and 27 alpha-helix; 40 and 38 beta-structure; and 41 and 35 aperiodic, respectively. Fluorescence emission max. showed a red shift from 330 nm, indicating structural alterations in the proteins due to ABT.

Functional properties of the total proteins of sunflower (Helianthus annuus L.) seed-effect of physical and chemical treatments

Changes in the functional properties, including N solubility, bulk density, water and fat absorption capacities, foaming capacity and stability and emulsion capacity, of sunflower seed proteins as a result of chemical (washing with acidic butanol) and physical (wet or dry heating) treatments were studied. Functional properties were evaluated in water and in 1M NaCl. Autoclaving at 2 kg/cm2 and roasting at 100 or 150C reduced the N solubility of the proteins. Acidic butanol treatment decreased the foam stability of proteins in water. Foam from samples roasted at 150C or autoclaved (1 or 2 kg/cm2) collapsed faster than foam from samples roasted at 100C or controls. In all cases, the emulsifying stability of proteins was higher in water than in NaCl. Results are explained with respect to surface and physicochemical properties of sunflower seed proteins.

Sunflower Protein Concentrates and Isolates’ Low in Polyphenols and Phytate

ABSTRACTProtein concentrates and isolates from sunflower (Helianthus annuus L.), essentially free of polyphenols and/or low in phytate, were prepared from dehulled seeds by a sequential extraction procedure using organic solvents, such as hexane for defatting and acidic butanol for removing polyphenols. Phytate was removed by aqueous extraction and separation at acidic and/or alkaline conditions, depending on the relative solubility of the protein and phytate. Reduced‐phytate concentrates and isolates developed off‐colors unless the polyphenol concentration was less than 0.05%. The protein solubility profile of the reduced‐phytate products was much better than that of the defatted meal, especially below the isoelectric point.

Isolation and partial characterization of phytotoxic compounds from lantana (Lantana camara L.)

Phytotoxic compounds obtained from methanol extracts of field-grown lantana (Lantana camara L.) were quantified in terms of their inhibition of ryegrass (Lolium multiflorum Lam.) seed germination and/or seedling growth. Subsequent partition of the aqueous fraction (derived from drying the MeOH extract in vacuo and redissolving in distilled water) at various pHs with solvents of differing polarity indicated the phytotoxic compounds were both polar and slightly acidic. Thin-layer Chromatographic separation of the acidic butanol fraction in a solvent mixture of butanol, acetic acid, water (4∶1∶5, by volume) yielded an active fraction with anR f value of 0.89-1.0 that was inhibitory to both root and shoot growth of ryegrass. Two other fractions withR f values of 0.04-0.23 and 0.41-0.57 were inhibitory only to root but not shoot growth.

Uptake and Metabolism of [14C]Salicylic Acid in Lemna gibba G3

When the long-day plant Lemna gibba L., strain G3 is grown under continuous light on ammonium-free half-strength Hutner's medium (NH(4) (+)-free 0.5 H medium) there is virtually no flowering, but addition of 10 micromolar salicylic acid (SA) to the medium results in substantial flowering. Using this system, the uptake and metabolism of [(14)C]SA in L. gibba G3 has been examined. SA uptake is rapid and linear for at least the first 24 hours. After 30 minutes, nearly 90% of the radioactivity in the plants is present as free SA. Part of this is rapidly converted to one or more bound forms of SA that appear either in the acidic butanol fraction or in the aqueous residue, and after 12 hours an equilibrium is reached between the free and bound forms of SA. When plants receive SA for 6 days and then are switched to control medium, both the free and bound SA remain nearly constant for at least 5 days. However, there is virtually no transfer of SA from mother fronds to daughter fronds, indicating that the SA is apparently sequestered within the cell. Cell fractionation studies show that nearly 95% of the SA remains in the supernatant even after a 2-hour centrifugation at 300,000 g. Thus, it is unlikely that SA is confined within a specific organelle, but rather is probably secreted into the vacuole.

Reversed-Phase High-Performance Liquid Chromatographic Analysis of Liothyronine Sodium and Levothyroxine Sodium in Tablet Formulations: Preliminary Studies on Dissolution and Content Uniformity

Levothyroxine sodium was estimated from tablet formulations of levothyroxine sodium and liotrix (liothyronine sodium-levothyroxine sodium combination tablet). The procedure consisted of the addition of 3,3′,5′-triiodothyronine as the internal standard to the pulverized sample, followed by an acidic butanol extraction, evaporation, and injection onto a μBondapak reversed-phase high-performance liquid chromatographic column. The eluent was methanol-water-phosphoric acid (50:50:0.1), and the effluent was monitored by UV detection at 254 nm. A standard linear calibration curve was obtained for direct standard solutions equivalent to 18-225 μg of levothyroxine sodium/tablet. The procedure is sensitive enough for single-tablet analysis. Using this procedure, content uniformity studies were performed on liothyronine sodium tablets, levothyroxine sodium tablets, and liotrix tablets. The procedure also was adapted for conducting dissolution studies on levothyroxine sodium tablets in deionized water using the rotating-paddle method.

EXTRACTION OF GOSSYPOL AND OLIGOSACCHARIDES FROM OILSEED MEALS

ABSTRACTRemoval of gossypol from cottonseed meal and fermentable sugars from soybean meal by butanol‐HCl solution was tested. Acidic butanol treatment yields low gossypol cottonseed protein concentrates and low oligosaccharides soy protein concentrates, respectively. This procedure does not appreciably alter proteins, as it is shown by the comparison of electrophoretic patterns of albumins and globulins extracted from meal and corresponding protein concentrates.

Acidic butanol removal of color-forming phenols from sunflower meal

Acidic butanol removal of color-forming phenols from sunflower meal

Diffusible and Extractable Gibberellins in Bean Cotyledons in Relation to Dwarfisni

AbstractDwarfism in bean plants (Phaseolus vulgaris L.) is investigated in relation to the diffusible and extractable gibberel‐lins in cotyledonary tissues. These gibberellins were partitioned into four parts prior to thin layer chromatograplty: non‐acidic and acidic ethyl acetate fractions, and non‐acidic and acidic butanol fractions.Cotyledonary segments from a tall plant (cv. Kentucky Wonder) seem to diffuse, preferentially acropetally, more gibberellins in each fraction than those from a dwarf plant (cv. Masterpiece). The diffusion increases with the length of the segments and decreases with period after sowing. From experiments on extraetable gibberellins, however, it is concluded that these phenomena actually result from differences in the gibbereliin contents of the tall and the dwarf plants, from differences in the gibberellin contents of the distal and proximal parts of cotyledons, and from the gibberellin contents before and after imbibition.Ten kinds of gibberellin‐Iike substances are detectable in the diffusates, exudates and extracts from the cotyledons of both the plants; two in the non‐acidic ethyl acetate, three in the acidic ethyl acetate (GA1, GA6 and another), two in the non‐acidic butanol, and three in the acidic butanol frac—tion. They are almost identical in quality in the dwarf and tall plants, but in the latter they are more abundant in the cotyledons, particularly in their distal part.With respect to the change in content during the period after sowing, the gibberellin‐Iike substances are classified in three groups; unchanging, decreasing (GA1, GA6, others) and increasing (butanol soluble glucosyl esters and glucosides of gibberellins)‐. The increase of glucose‐bound gibberellins and the decrease of free gibberellins during the sowing period suggest the occurrence of conversion. This is obvious only in the tall plants. The gibberellin content in cotyledons is higher in the tall plant than in the dwarf plant. Thus, the marked hypocotyl growth in the tall plants may be dependent on the higher content of gibberellins in their cotyledons and on the higner rate of conversion from free to bound forms.

The nature of tannin in rapeseed ( Brassica campestris)

Polyphenols were present in the seed coats or testas of rapeseed. Although some oligomeric phenol was found in aqueous extracts of defatted seeds, most of the polymeric material was apparently strongly bound to other structures or constituents in the seed coat. Digestion of the seed coats with hot acidic butanol liberated alcohol-soluble pigments which were identified as anthocyanidins. It may be concluded from these results that a condensed tannin occurs in rapeseed.

Seasonal Changes in Growth Promoters and Inhibitors in Buds of Apricot1

Abstract Spur buds of apricot were sampled at intervals from late July, 1968, until bloom in 1969, and from 1 month after bloom until the end of September. The samples were freeze-dried and then extracted with 80% methanol. The extracts were partitioned into 4 phases: water, neutral ethyl acetate, acidic ether, and acidic butanol, and then bioassayed for auxins, inhibitors and gibberellins. Although an inhibitor and gibberellin-like activity were present in the extracts, no consistent auxin-type activity was found. The end of rest was correlated with a decrease in the level of inhibitor and gibberellin-like activity, with the inhibitor increasing again towards anthesis. The onset of rest in May, 1969, was correlated with a decrease in inhibitor and increase in gibberellin-like activity. The inhibitor was present in scales and floral parts of buds, while gibberellin-like activity was confined mainly to the floral parts.

Changes in Endogenous Growth Substances in the Embryos of Juglans regia During Stratification1

Abstract Kernels from Juglans regia walnuts stratified at 0°C were sampled at weekly intervals and extracted with methanol. The extracts were partitioned into 4 phases which were water, neutral ether, acidic ether and acidic butanol, then bioassayed for cytokinins, gibberellins, auxins and inhibitors. No cytokinins nor gibberellins were found in the tissue. There was activity analogous to that from auxins. An inhibitor which diminished during stratification was found. This inhibitor is believed to be abscisic acid, on the basis of UV absorption spectrum, Rf values established by co-chromatography on paper and silica gel plates, and derivatives analyzed by gas liquid chromatography.

Variations in Endogenous Gibberellins in Developing Bean Seeds II. Changes Induced in Acidic and Neutral Fractions by GA1

Immature (8-mm), medium mature (11-mm), and mature green (16- and 17-mm) bean seeds (Phaseolus vulgaris L. cv. Kentucky Wonder and Bountiful) were incubated in gibberellin A(1) solutions for 24 hours at 20 degrees . Extracts from the seeds were separated into nonacidic, acidic ethyl acetate, and acidic butanol fractions. These were chromatographed. The eluates of the chromatograms were tested on Progress No. 9 dwarf peas grown under red light. The level of neutral gibberellin-like substances remained unchanged in immature seed, but they increased markedly in mature green seeds. Coincident with increased levels of the neutral substances, there were significant decreases in acidic ethyl acetate-soluble gibberellin-like substances, including applied GA(1), and in 1 acidic butanol-soluble gibberellin-like substance. Seed incubation in GA(1) brought about increased activity of substance B-II in immature and medium mature seeds. The level of butanol-soluble gibberellin-like substance B-I in seeds of any size was not affected by incubation in GA(1). Considering the marked increases in activity induced in the neutral fraction and the decreases in activity of certain eluates from the chromatograms of the acidic fractions, it was concluded that the neutral fraction may serve as a reserve form of gibberellins in the dry seed. The acidic ethyl acetate substances and substance B-II may be required for normal development of the bean seed.

Variations in Endogenous Gibberellins in Developing Bean Seeds I. Occurrence of Neutral and Acidic Substances

Activities of separated and chromatographed substances in the nonacidic, acidic ethyl acetate and acidic butanol fractions from bean seeds, Phaseolus vulgaris L., cv. Bountiful and Kentucky Wonder, were measured in the Progress No. 9 dwarf pea bioassay grown under red light. Activity in the nonacidic fraction was shown to be attributable only to neutral substances and was free of acidic gibberellin-like substances. As the seed matures, neutral substances and one of the acidic butanol-soluble substances (B-I) increase in activity. The acidic ethyl acetate substances and butanol-soluble substance (B-II) initially increase and then almost disappear.