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Fluorescence densitometric method for the determination of gluconic and lactobionic acids ("sugar acids") in pharmaceutical preparations.

An in situ fluorimetric method has been developed for the quantitation of gluconic and lactobionic acids and their salts in tablet formulations. The method is based on glycol cleavage with lead tetraacetate followed by treatment with dichlorofluorescein. Calcium gluconate and lactobionate were determined in Calcium-Sandoz and Ca-C 1000 Sandoz effervescent tablets. The reproducibility corresponded to relative standard deviations between 0.7 and 3.5% (usually below 2%). Detection limits of 0.2 mug per spot can be obtained. Interfering compounds such as citric acid, sugars and ascorbic acid can be separated from the "sugar acids". The linearity of the calibration graphs between 0.5 and 5 mug per spot is satisfactory (r = 0.994-0.999). The method is simple and could be applied to the routine analysis of suitable pharmaceutical formulations. Other compounds with glycol structures should also be adaptable to this technique.

Chromatography, Thin Layer

[Preparation and analysis of hydroxycarboxylic acid, sugar acid and amino acid complexes of gallium(III)].

Neutral, stable in water and not hydrolysing gallium(III) complexes were prepared and analysed by instrumental analytical methods. Nine hydroxy-carboxylic acids, four sugar acids and four amino acids were used as ligands. The chemical and physical characteristics of complexes (composition, stability, water solubility, pH in aqueous solution) significantly depend on the quality of gallium salt and ligand, the ratio of gallium and ligand, the medium used during the preparation (pH and temperature).

Amino Acids

Determination of carboxylic acids, sugars, glycerol and ethanol in wine and grape must by ion-exchange high-performance liquid chromatography with refractive index detection.

Method to determine the major carboxylic acids, sugars, glycerol and ethanol in wine and grape must was developed using an ion-exchange column and refractive index detector. A solid-phase extraction method with a strong anion exchanger was used to determine these compounds in sweet wines and in grape musts. With this method it is possible to determine malic acid in sweet wines and in grape musts without interference from sugars. This high-performance liquid chromatographic method was compared with standard methods of analysis. There was good agreement in the accuracy and precision of the compared methods.

Carbohydrates

The effect of endogenous sugar acids on the afferent discharge rate of the hepatic branch of the vagus nerve in the rat.

The effects of intraportal administration of endogenous sugar acids such as 2DTA, 3DPA and 2B40 on afferent activity of the hepatic branch of the vagus nerve were investigated in the rat. A decrease in afferent discharge rate was observed after administration of 2DTA (0.1 mM approximately 10 mM, 0.1 ml) and 2B4O (100 nM approximately 2 microM, 0.1 approximately 0.2 ml), whereas 3DPA (0.1 mM approximately 10 mM, 0.1 ml) increased the afferent activity. These observations suggest that 2DTA and 2B4O suppress, and 3DPA enhances food intake behavior and may be important in the regulation of blood glucose homeostasis through glucose-sensitive vagal hepatic afferent fibers.

4-Butyrolactone

Structural and stereoisomeric specificity of serum-borne sugar acids related to feeding control by rats.

Specificity of chemical structures and stereoisomers among serum-borne short-chain organic acids in rats were assessed for their effects on feeding behavior and humoral factors by infusion into the rat third cerebroventricle. Infusion of glyceric acid (1.0 mumol), 3,4-dihydroxybutanoic acid gamma-lactone (3,4-DB) or 3,4,5-trihydroxypentanoic acid gamma-lactone (2.50 mumol) immediately before the dark phase decreased food intake for, at most, 24 h. These acids did not significantly affect drinking or ambulation. Initial feeding, not necessarily accompanied by periprandial drinking, was induced after infusion of 2,4-dihydroxybutanoic acid gamma-lactone, 2,4,5-trihydroxypentanoic acid gamma-lactone (2,4,5-TP) or exogenous 2,4,5,6-tetrahydroxyhexanoic acid gamma-lactone (2.50 mumol) in the light phase. Of these acids, 3,4-DB most potently suppressed and 2,4,5-TP most potently enhanced feeding. Of these, the 2S,4S-isomer and the 3S-isomer were the most potent of 2,4,5-TP and 3,4-DB, respectively. Only the 2S,4S-isomer of 2,4,5-TP induced hypoglycemia with hyperinsulinemia, whereas opposite effects were produced by the 3S-isomer of 3,4-DB. The results indicate that the positions of the hydroxyl groups on 4-butanolide and the S- and S,S-stereoisomers are important in modulating food intake through the hypothalamus.

Animals

Structural characteristics of endogenous sugar acids and relations to feeding modulation.

Structural specificity among short-chain organic acids for effects on feeding behavior, blood glucose and insulin was investigated by infusion of 1 exogenous and 6 endogenous derivatives into the rat third cerebral ventricle. Glyceric acid (GEA) (1.0 mumol), 3,4-dihydroxybutanoic acid gamma-lactone (3,4-DB) and 3,4,5-trihydroxypentanoic acid gamma-lactone (3,4,5-TP) (2.50 mumol) decreased food intake for, at most, 24 h. These acids depressed the size of the first meal after infusion, but did not affect latency to the first meal, eating speed, drinking or ambulation. Infusion of 2,4-dihydroxybutanoic acid gamma-lactone (2,4-DB) (1.25 mumol), 2,4,5-trihydroxypentanoic acid gamma-lactone (2,4,5-TP), and an exogenous compound, 2,4,5,6-tetrahydroxyhexanoic acid gamma-lactone (2,4,5,6-TH) (2.50 mumol), induced transient initial feeding which was not necessarily accompanied by periprandial drinking. Ambulation was concomitantly increased. Of these organic acids, 3,4-DB and 2,4,5-TP were most potent in their effects on feeding. Hyperglycemia was induced by 2.50 mumol 3,4-DB leaving insulin unaffected; 2.50 mumol 2,4,5-TP caused hypoglycemia, with a persistent but not significant rise in insulin. The results suggest that slight structural differences of endogenous organic acids, in particular the positions of hydroxyl groups on the lactone ring of 4-butanolide, may be important in feeding modulation by conveying intrinsically reciprocal signals to neurons involved in feeding and satiety.

Animals

Preferences by northern grasshopper mice for solutions of sugars, acids, and salts in Richter-type drinking tests.

Taste preferences, as measured in 48-hour, Richter-type drinking tests (test solution opposite distilled water), were determined for northern grasshopper mice (Onychomys leucogaster, ssp. breviauritus). The Ss were nine males and nine females which were individually housed within an environmental chamber. The test solutions were prepared from five sugars (fructose, glucose, lactose, maltose, sucrose) three salts (magnesium sulfate, potassium chloride, sodium chloride), and two acids (citric acid and hydrochloric acid). In randomly assigned order, each sugar and each salt solution was presented at five molar concentrations, and each acid was paired with distilled water at six levels of pH. Strong drinking preferences were shown for all concentrations of the sugars above .05-.10 M, and sugars ranked in order of preference as follows: Maltose = sucrose greater than glucose = fructose greater than lactose. Preferences were also shown for hypotonic concentrations of NaCl. The other salts and both acids, however, were indifferently preferred at low concentrations and were rejected at the higher concentrations. Taste preferences by grasshopper mice for these chemicals were similar to those exhibited by Monogolian gerbils tested with the same items. The similar patterns of preference shown by New World cricetid rodents (grasshopper mice) and Old World cricetid rodents (gerbils) suggest that conclusions concerning disparity in taste sensibilities among animal forms may be premature.

Acids

4-O-(1-carboxyethyl)-L-rhamnose, a second unique acidic sugar found in an extracellular polysaccharide from Butyrivibrio fibrisolvens strain 49.

Butyrivibrio fibrisolvens strain 49 excretes a polysaccharide that contains D-glucose, D-galactose, 4-O-(1-carboxyethyl)-D-galactose, and an acidic component of previously unknown structure. We report here the identity of the unknown as 4-O-(1-carboxyethyl)-L-rhamnose. The structure of this previously unknown compound was deduced from (1) comprehensive electron-impact and chemical-ionization mass-spectroscopic studies of differentially labelled derivatives prepared from the unknown, (2) 13C-n.m.r. and 1H-n.m.r. studies of purified neutral sugars derived from the unknown and (3) chemical degradation experiments.

Chemical Phenomena

Central action of endogenous sugar acid (2-buten-4-olide): comparison with local anesthesia in hypothalamus.

Rats were trained to discriminate cue tone stimuli (CTS) predicting reward (CTS+) [juice or intracranial self-stimulation (ICSS)], or aversion (CTS-) (mild electric shock or tail pinch). Unit activity in the lateal hypothalamus (LHA) and lateral preoptic-anterior hypothalamic area (lPOA-AHA) of the rat was recorded during CTS learning. The effects of local anesthesia of the amygdala (AM), ventral tegmental area (VTA) or LHA by procaine hydrochloride, and the effects of intraperitoneal or intravenous 2-buten-4-olide (2-B4O) on LHA neural activity and licking behavior were compared. LHA neurons differentiated between rewarding and aversive stimuli, and acquired corresponding discrimination of CTS+ and CTS-. In the lPOA-AHA, neurons responded similarly to CTS+, rewarding stimuli, CTS- and aversive stimuli. Procainization of the AM suppressed LHA neural responses to CTS1+ predicting juice, and stopped licking for juice. Procainization of the VTA suppressed LHA neural responses to CTS2+ predicting ICSS, and stopped licking for ICSS. LHA procainization suppressed both licking for juice and ICSS. Both intraperitoneal and intravenous 2-B4O stopped licking for juice and ICSS, but did not influence LHA responses to CTS1+ or CTS2+. The results suggest that dynamic interaction of AM-LHA-VTA are important for CTS+ learning, and 2-B4O acts directly on LHA neurons while maintaining afferent sensory inputs to the LHA.

4-Butyrolactone

Transcriptional landscape and dynamics involved in sugar and acid accumulation during apple fruit development.

In fleshy fruit, sugars and acids are central components of fruit flavor and quality. To date, the mechanisms underlying transcriptional regulation of sugar and acid during fruit development remain largely unknown. Here, we combined ATAC-seq with RNA-seq to investigate the genome-wide chromatin accessibility and to identify putative transcription factors related to sugar and acid accumulation during apple (Malus domestica) fruit development. By integrating the differentially accessible regions and differentially expressed genes, we generated a global data set of promoter-accessibility and expression-increased genes. Using this strategy, we constructed a transcriptional regulatory network enabling screening for key transcription factors and target genes involved in sugar and acid accumulation. Among these transcription factors, 5 fruit-specific DNA binding with one finger genes were selected to confirm their regulatory effects, and our results showed that they could affect sugar or acid concentration by regulating the expression of sugar or acid metabolism-related genes in apple fruits. Our transcriptional regulatory network provides a suitable platform to identify candidate genes that control sugar and acid accumulation. Meanwhile, our data set will aid in analyzing other characteristics of apple fruit that have not been illuminated previously. Overall, these findings support a better understanding of the regulatory dynamics during apple fruit development and lay a foundation for quality improvement of apple.

Malus

Chemical composition of Litomosoides carinii microfilarial sheaths.

Litomosoides carinii microfilariae were exsheathed by freezing and thawing, and the sheaths were separated by filtration. Samples of pure sheaths thus obtained were hydrolyzed, methanolyzed or oxidized with nitric acid under pressure at 300 degrees C, respectively, and were analyzed for amino acids, sugars, fatty acids or for metal ions and phosphorus. Almost 75% of the sheath dry weight could thus be accounted for. Amino acids (55 weight %) were the major constituents, and amongst these glutamine and proline (approximately 11% each). The detection of 2% cysteine/cystine indicated the possible presence of disulfide crosslinks. Besides amino acids, approximately 8% of sugars--roughly equimolar amounts of (N-acetyl)galactosamine and uronic acids--1.5% of monovalent cations (Na+ and K+) and 9.5% of phosphate were detected. No appreciable amounts of fatty acids, neutral sugars, neuraminic acid, or (N-acetyl)glucosamine (i.e. no chitin) were found.

Amino Acids

Developmental aspects of maleic acid-induced inhibition of sugar and amino acid transport in the rat renal tubule.

The transport of alpha-aminoisobutyric acid and alpha-methyl-d-glucoside by isolated tubules from Sprague-Dawley rats at different stages of development follows a separate age-dependent pattern for each substrate. The effects of 6 M maleic acid on transport processes differ for amino acids and sugars and become manifest at distinct points during development. Maximum inhibition by maleic acid occurs at a time subsequent to maturity of these transport systems. In an effort to explain these transport phenomena, the uptake and metabolism of 14C-labeled maleic acid by the newborn or adult renal tubule was studied, showing significant binding by the tubule membrane, penetration of the cell by diffusion, and no conversion to 14CO2. Maleic acid has no demonstrable effect on the membrane-associated enzymes which are thought to play a role in the transport of small molecules.

Age Factors

High-performance liquid chromatographic investigation of the amino acid, amino sugar and neutral sugar content in glycoproteins.

A methods for the simultaneous separation and determination of amino acids, amino sugars and neutral carbohydrates is described. Stepwise elution systems with sodium citrate and borate buffers have developed for the ion-exchange liquid chromatographic separation of amino acids and sugars, using 8-micrometer particle size resins and the Stein and Moore and orcinol colorimetric method for detection. With the aid of this system, the direct quantitative comparison of sugars and amino acids by liquid chromatography becomes possible for the first time.

Amino Acids

Effects of Mn-deficit feeding to ewes on certain amino acids and sugars in cartilage of their newborn lambs.

The distribution of amino acid, amino sugar, and uronic acid in the epiphyseal cartilage of newborn lambs were examined to determine the influence of manganese (Mn) deficiency. Throughout the gestational period, one group (group I) of eight pregnant ewes was given a ration containing 60 mg of Mn/kg of feed and a second group (group II) of eight ewes was given a ration containing 5 mg of Mn/kg of feed. The neonatal lambs were killed, and the epiphyseal plates from various bones in the thoracic limb were removed and analyzed for glucosamine, galactosamine, uronic acid, and 17 amino acids. Concentrations of uronic acid and of the sulphur amino acids (methionine not significant, P greater than 0.05) were larger in the cartilage from the lambs born of group I ewes (60 mg of Mn/kg of ration) than in the cartilage from those of the Group II ewes (Mn deficiency). A tendency for higher hexosamine values was observed in the organic matrix of the group I lambs than of the group II lambs. However, concentrations of serine, histidine, and lysine were higher in the cartilage from group II lambs (Mn deficiency). Thus, the role of Mn in the prevention of joint abnormalities may be its influence on the mucopolysaccharide and protein composition of the epiphyseal plate.

Amino Acids

Synthesis of sialic acid-containing nucleotide sugars: CMP-sialic acid analogs.

Syntheses of some sialic acid-containing nucleotide sugars are reported. The reaction of methyl[(2-hydroxy)ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galacto -2- nonulopyranosid]onate (4) with various fully protected hydrogen phosphonates of nucleotides (5a-c) in the presence of 2,4,6-triisopropylbenzenesulfonyl chloride (TPS-Cl), gave, after oxidation and deprotection, the corresponding sialic acid-containing nucleotide sugar analogs (8a-c).

Cytidine Monophosphate N-Acetylneuraminic Acid

Synthesis of sialic acid-containing nucleotide sugars: CMP-sialic acid analogs.

Syntheses of some sialic acid-containing nucleotide sugars are reported. The reaction of methyl [(2-hydroxy)ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galacto -2-nonulopyranosid]onate (4) with various fully protected hydrogen phosphonates of nucleosides (5a-c) in the presence of 2,4,6-triisopropylbenzenesulfonyl chloride (TPSCl), gave, after oxidation and deprotection, the corresponding sialic acid-containing nucleotide sugar analogs (8a-c).

Antiviral Agents