The estimation of acid glycosaminoglycan-Alcian blue complexes eluted from electrophoretic strips

The present report describes a new and simple method for the detection and quantitative determination of pentoses and hexuronic acids either separately or in a mixture. The method involves treatment of the sample with sulfuric acid followed by the addition of harmine and then cysteine. Hexuronic acids produce a pink color with harmine, while pentoses react only after the addition of cysteine. Hexosamines, methylpentoses, and moderate amounts of hexoses do not interfere with these determinations.The method is simple, accurate, and allows the determination of small amounts of pentoses in the presence of larger amounts of hexuronic acids and other sugars.     

A rapid method for seperation and identification of several hexuronic acids and hexuronic acid-containing oligosaccharides.

Five naturally occurring hexuronic acids and several hexuronic acid-containing oligosaccharides were separated and identified by high voltage paper electrophoresis, using one of the following buffers. (i) Pyridine-acetic acid-water (1:10:89, by volume), the pH of which was adjusted to 2.3–3.5 with 98% formic acid. (ii) Pyridine-water (1:90, by volume), the pH of which was adjusted to 3.5–4.0 with glacial acetic acid. The best separation of the five hexuronic acids and heparin disaccharides was observed at pH 2.7 after electrophoresis for 180 min at 100 V/cm. At pH 3 l-gulosyluronic acid-l-guluronic acid could be easily isolated from an acid hydrolysate of alginate by the present method.     

G.L.C. of the O-trimethylsilyl derivatives of hexuronic acids

The free acids, sodium salts, and lactones of several hexuronic acids have been studied as their O-trimethylsilyl derivatives by gas-liquid chromatography using SE-30 and XE-60 liquid phases. Silylation was best performed in methyl sulphoxide. The equilibrium between the various forms of a hexuronic acid in methyl sulphoxide was also studied by g.l.c. following silylation. The hexamethyldisilazane used in the silylation disturbed the equilibrium attained in the solvent, but this was overcome by premixing the hexamethyldisilazane with chlorotrimethylsilane. Methyl sulphoxide and the silylating reagents gave a two-phase system in which the derivative was favourably partitioned into the upper layer. Partition coefficients and stabilities of the derivatives were measured, and a g.l.c. method for the analysis of the hexuronic acids was thereby developed. The oximes of the hexuronic acids were studied as alternative derivatives for g.l.c., and their equilibrium compositions and g.l.c. retention times are recorded.     

Stereospecific synthesis and two-dimensional 1H-NMR investigation of isoursocholic acid.

This report describes the chemical synthesis of isoursocholic acid (3 beta, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoic acid) from its corresponding 3 alpha-analog. The method consists of refluxing a mixture of ursocholic acid, triphenylphosphine, and diethyl azodicarboxylate in benzene solution with an acid such as formic acid. The sterically pure ester (3 beta-formate) so formed after saponification with LiOH-aqueous methanol then allowed an easy access to the epimer of the starting acid. Large scale preparative separation and purification of the final product and synthetic intermediates were accomplished by flash column chromatography of their methyl ester derivatives. Structural assignment of the isourscholic acid molecule was confirmed by complete analysis of proton NMR spectra using 2-D NMR correlation experiments which rigorously established the (3 beta/3 alpha) and (7 beta/7 alpha) hydroxyl configurations in the isoursocholic and ursocholic acids. It is suggested that the isoursocholic acid will be useful as a reference compound and as a substrate in studies dealing with the hepatic inversion of the 3 beta-hydroxy group.     

Mass-spectral fragmentation of methyl 2,3,4-tri-O-methyl-α-D-glucopyranosiduronamide and its analytical application

Fragmentation pathways for methyl 2,3,4-tri-O-methyl-α-D-glucopyranosiduronamide are proposed, based on 70- and 12-eV spectra of the compound specifically labelled with CD₃ and ND₂ groups. The presence of the NH₂ group in the molecule gives rise to new fragmentation series. The number and positions of CD₃ groups can be unequivocally determined from the mass spectra. Partially methylated derivatives of hexuronic acids, obtained by methylation analysis of hexuronic acid-containing substances, can be identified by exhaustive trideuteriomethylation and conversion into readily obtainable crystalline amides.     

Effects of ursodeoxycholic acid, analogues of ursodeoxycholic acid and combination of bile acids on bile acid synthesis in cultured rat hepatocytes

The effect of individual 7 beta-hydroxy bile acids (ursodeoxycholic and ursocholic acid), bile acid analogues of ursodeoxycholic acid, combination of bile acids (taurochenodeoxycholate and taurocholate), and mixtures of bile acids, phospholipids and cholesterol in proportions found in rat bile, on bile acids synthesis was studied in cultured rat hepatocytes. Individual steroids tested included ursodeoxycholate (UDCA), ursocholate (UCA), glycoursodeoxycholate (GUDCA) and tauroursodeoxycholate (TUDCA). Analogues of UDCA (7-methylursodeoxycholate, sarcosylursodeoxycholate and ursooxazoline) and allochenodeoxycholate, a representative of 5 alpha-cholanoic bile acid were also tested in order to determine the specificity of the bile acid biofeedback. Each individual steroid was added to the culture media at concentrations ranging from 10 to 200 microM. Mixtures of taurochenodeoxycholate (TDCA) and taurocholate in concentrations ranging from 150 to 600 microM alone and in combination with phosphatidylcholine (10-125 microM) and cholesterol (3-13 microM) were also tested for their effects on bile acid synthesis. Rates of bile acid synthesis were determined as the conversion of added lipoprotein [4-14C]cholesterol or [2-14C]mevalonate into 14C-labeled bile acids and by GLC quantitation of bile acids secreted into the culture media. Individual bile acids, bile acid analogues, combination of bile acids and mixture of bile acids with phosphatidylcholine and cholesterol failed to inhibit bile acid synthesis in cultured hepatocytes. The addition of UDCA or UCA to the culture medium resulted in a marked increase in the intracellular level of both bile acids, and in the case of UDCA there was a 4-fold increase in beta-muricholate. These results demonstrate effective uptake and metabolism of these bile acids by the rat hepatocytes. UDCA, UCA, TUDCA and GUDCA also failed to inhibit cholesterol-7 alpha-hydroxylase activity in microsomes prepared from cholestyramine-fed rats. The current data confirm and extend our previous observations that, under conditions employed, neither single bile acid nor a mixture of bile acids with or without phosphatidylcholine and cholesterol inhibits bile acid synthesis in primary rat hepatocyte cultures. We postulate that mechanisms other than a direct effect of bile acids on cholesterol-7 alpha-hydroxylase might play a role in the regulation of bile acid synthesis.     

Synthesis of glucuronoglucans in chicken cartilage cultured in synthetic medium.

Cartilaginous femur and tibia rudiments from 10-day-old chick embryos were grown in vitro for 4 days in Parker's solution without protein added, and subsequently fixed and extracted successively with 0-2 N HClO4 at 4 degrees C (fraction A), 5 per cent trichloracetic acid (TCA) at 4 degrees C (fraction B), and 5 per cent TCA at 90 degrees C (fraction C). The residue after extraction was dissolved in 1 N NaOH at room temperature (fraction D). Fraction C containing most of hexuronic acids and aminosugars of the cartilage was used to study the quantitative changes of glucuronoglucans throughout the culture period. The amount of hexuronic acids and aminosugars was increased after 24, 48, 72 and 96 hours of culture. After 96 hours the level of hexuronic acids was twice that found prior to establishing the culture. The increment was statistically significant.     

Improved analytical method for the hexuronic acids at the reducing terminals of glycosaminoglycans

When endo-uronidases act on glycosaminoglycans, the reaction products have hexuronic acid residues at the reducing terminals. An analytical method for hexuronic acids at the reducing terminals was devised for hyaluronate oligosaccharides having hexuronic acid residues at the reducing terminals.The procedure is as follows: Hexuronic acid residues at the reducing terminals of hyaluronate oligosaccharides were tritiated with reduction using NaB[³H]₄ and the products were hydrolyzed with trifluoroacetic acid and nitrous acid. As a result, the tritiated and reduced hexuronic acid residues, that is aldonic acids, were liberated from the reducing terminals. After passing them through anion and cation ion-exchange resins, the aldonic acids were lactonized. The lactones were developed on paper chromatography, and their radioactivities determined on the paper.The method is also useful for discrimination between glucoronic acid and iduronic acid at the reducing terminals of glycosaminoglycans.     

Hexuronic acid dehydrogenase of Agrobacterium tumefaciens

Growth of Agrobacterium tumefaciens on d-glucuronic acid (GlcUA) or d-galacturonic acid (GalUA) induces formation of hexuronic acid dehydrogenase [d-aldohexuronic acid: nicotinamide adenine dinucleotide (NAD) oxidoreductase]. The dehydrogenase, which irreversibly converts GlcUA or GalUA to the corresponding hexaric acid with the concomitant reduction of NAD, but not of nicotinamide adenine dinucleotide phosphate was purified 60-fold by MnCl(2) treatment, (NH(4))(2)SO(4) fractionation, chromatography on diethylaminoethyl Sephadex and negative adsorption with Ca(3)(PO(4))(2) gel. The pH optimum is 8.0. Other uronic acids, aldohexoses, aldopentoses, and polyols, are not substrates. Reduced nicotinamide adenine dinucleotide is an inhibitor strictly competitive with NAD. Kinetic data indicate that the dehydrogenase induced by growth on GlcUA may not be identical with that induced by growth on GalUA.     

Identification of uronic acid oxidase in plant peroxidase preparations

Commercial plant peroxidase preparations contained a uronic acid oxidase, separable from the peroxidase activity by ion exchange chromatography. The partially purified enzyme, devoid of peroxidase, oxidized hexuronic acids, with the greatest activity for D-glucuronic acid, whereas other aldoses were not substrates. The immediate products of reaction of D-glucuronic acid with oxygen were hydrogen peroxide and a D-glucarolactone, which was a very strong inhibitor of β-glucuronidase and believed to be the 1,5-lactone. The sensitivity to sulphite inhibition suggests that the enzyme is a flavoprotein.     

Novel azido fatty acid ester derivatives from conjugated C(18) enynoate.

Methyl octadec-11Z-en-9-ynoate (1) was epoxidized to give methyl 11,12-Z-epoxy-octadec-9-ynoate (2, 81%). Acid catalyzed ring opening of the epoxy ring of compound 2 gave methyl 11,12-dihydroxy-octadec-9-ynoate (3, 80%). The latter was treated with mesyl chloride to yield methyl 11,12-dimesyloxy-octadec-9-ynoate (4, 76%). Reaction of compound 4 with sodium azide furnished methyl 11-azido-12-mesyloxy-octadec-9-ynoate (5a, 49%) and methyl 11-azido-octadec-11E-en-9-ynoate (5b, 24%). Compound 2 was semi-hydrogenated over Lindlar catalyst to give methyl 11,12-Z-epoxy-octadec-9Z-enoate (6, 90%). This allylic epoxy fatty ester (6) was reacted with sodium azide to give a mixture of methyl 11-azido-12-hydroxy-octadec-9Z-enoate (7a) and methyl 9-azido-12-hydroxy-octadec-9E-enoate (7b), which could not be separated into individual components by silica chromatography. Chromic acid oxidation of the mixture of compounds 7a and 7b furnished methyl 9-azido-12-oxo-octadec-10E-enoate (8, 42% based on amount of compound 6 used) and an intractable mixture of polar compounds. The various products were characterized by NMR spectroscopic and mass spectral analyses.     

Reverse cross-coupling in the synthesis 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid.

The present report describes the characterization of (24R and 24S)-27-nor-24-methyl-3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acids obtained in considerable amounts during the synthesis of (25RS)-3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acid by the electrolytic coupling of chenodeoxycholic acid and the half ester of methylsuccinic acid. The mixture of 24R and 24S diastereomers was resolved by analytical and preparative thin-layer chromatography and characterized by gas-liquid chromatography, proton magnetic resonance, and molecular rotation differences. For reference, the model compound, 27-nor-3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acid, was synthesized by electrolytic coupling of chenodeoxycholic acid and the half ester of succinic acid.     

Presumed role of mucilage of plantain seeds in spread of tobacco mosaic virus

A strain of tobaoco mosaic virus(TMVbs) isolated fromPlantago major L. is not seed-borne with this plant species; however, plantain seeds and the mucilage on their surface also contain the virus. The mucilage shows a very low infectivity; the visoous mucilage inhibits infeotion in rubbed leaves both in a mixture with the virus and if applied before (but not after) the inoculation of the virus. Polysaccharides and hexuronic acids were detected in the great plantain seed mucilage. A mixture of mucilage and viras stored at room temperature showed s low infectivity until 33 days, but the infectivity increased oonspicuously on the 40th day, apparently simultaneously with the decay of mucilage. Polysaccharides are suggested as being responsible for the inhibition. They affect the inoculated leaf but not the virus, as ascertained by eleotron mioroscopy and serology. The seed mucilage being an important factor in the spread of plantain seeds may act as a vehicle of the virus as well.     

Application of mixtures of tartaric acid derivatives in resolution via supercritical fluid extraction

Racemic N-methylamphetamine (rac-MA) was resolved with 2R,3R-tartaric acid (TA) and its derivatives (O,O'-dibenzoyl-(2R,3R)-tartaric acid monohydrate (DBTA) and O,O'-di-p-toluoyl-(2R,3R)-tartaric acid (DPTTA)), individually and using them in different combinations. After partial diastereomeric salt formation, the free enantiomers were extracted by supercritical fluid extraction using carbon dioxide as solvent. DBTA and DPTTA are efficient resolving agents for rac-MA, the best chiral separation being obtained at a molar ratio of 0.25 resolving agent to racemic compound for both resolving agents (ee(E) = 82.5% and ee(E) = 57.9%, respectively). Compared with the two other acids, TA is practically unsuitable for enantiomer separation (ee(E) < 5%). Applying a mixture of one individually active and one ineffective acid in half the equivalent molar ratio, when the acids are in 1:1 ratio in the mixture, the resolution efficiency values obtained exceeded those obtained by using the components individually. Decreasing the molar ratio of resolving agent mixture to 0.25, at which the individual resolving agents give the best chiral separation, the obtained resolution efficiency values did not differ significantly from those expected. The outcome of the resolution process depended only on the amount of the individually active resolving agents in the mixture.     

Ursodeoxycholic acid, chenodeoxycholic acid, and 7-ketolithocholic acid are primary bile acids of the guinea pig

Guinea pig gallbladder bile contains chenodeoxycholic acid (62 +/- 5%), ursodeoxycholic acid (8 +/- 5%), and 7-ketolithocholic acid (30 +/- 5%). All three bile acids became labeled to the same specific activity within 30 min after [3H]cholesterol was injected into bile fistula guinea pigs. When a mixture of [3H]ursodeoxycholic acid and [14C]chenodeoxycholic acid was infused into another bile fistula guinea pig, little 3H could be detected in either chenodeoxycholic acid or 7-ketolithocholic acid. But, 14C was efficiently incorporated into ursodeoxycholic and 7-ketolithocholic acids. Monohydroxylated bile acids make up 51% and ursodeoxycholic acid 38% of fecal bile acids. After 3 weeks of antibiotic therapy, lithocholic acid was reduced to 6% of the total, but ursodeoxycholic acid (5-11%) and 7-ketolithocholic (15-21%) acid persisted in bile. Lathosterol constituted 19% of skin sterols and was detected in the feces of an antibiotic-fed animal. After one bile fistula guinea pig suffered a partial biliary obstruction, ursodeoxycholic and 7-ketolithocholic acids increased to 46% and 22% of total bile acids, respectively. These results demonstrate that chenodeoxycholic acid, ursodeoxycholic acid, and 7-ketolithocholic acid can all be made in the liver of the guinea pig.     

Isolation of deoxyribonucleic acid and ribosomal ribonucleic acid from bacteria

1. Nucleic acids were released from Escherichia coli by lysing with tri-iso-propylnaphthalene sulphonate and 4-aminosalicylate and then extracting with a phenol-cresol mixture. 2. Nucleic acids were similarly released from Bacillus subtilis after initial treatment with lysozyme. 3. DNA was sedimented after careful precipitation with m-cresol or 2-butoxyethanol (0.1-0.12vol.) in the presence of 20% sodium benzoate. 4. Contaminating ribosomal RNA was removed by precipitation in the presence of 4m-sodium chloride or by extracting DNA with an acetate-butyrate mixture, in which RNA is insoluble. 5. The DNA from B. subtilis has a transforming ability of 0.3-0.6% for the tryptophan marker. 6. Ribosomal RNA was then precipitated with rapidly labelled RNA by the addition of an equal volume of 2-butoxyethanol. 7. There was good separation of the nucleic acids from protein and polysaccharides.     

A rapid vapor-phase acid (hydrochloric acid and trifluoroacetic acid) hydrolysis of peptide and protein

A new method for the acid hydrolysis of protein is presented. Peptide bonds are cleaved by the action of an HCl/trifluoroacetic acid (TFA) vapor mixture. Contamination for the hydrolysis mixture is reduced to low levels (1-3 pmol). Recovery of hydrophobic amino acid is improved. Short reaction times are achieved and rapid removal of acids is facilitated. The reaction temperature is 158 degrees C for reaction times of 22.5 and 45 min with 7 M HCl and 10% TFA containing 0.1% phenol.     

Acyl migration on nucleic acid compounds related to adenosine

Treatment of N6,N6-di-p-toluyl-2',3'-O-isopropylideneadenosine (7) with ZnBr2 in 1,4-dioxane afforded a 8,5'-O-cycloadenosine derivative 8 exclusively. Reaction of 2',3'-O-isopropylideneadenosine (1) with p-cyanobenzoyl chloride in a CH2Cl2-Et3N mixture afforded a ring-cleaved compound 11 as the main product.     

The relationship between Toluidine Blue staining and hexuronic acid content of cartilage matrix

Synopsis The relationship between the density of Toluidine Blue staining and the hexuronic acid content of xiphoid cartilage matrix has been investigated by microdensitometry and found to be linear. A given percentage reduction in the hexuronic acid content was accompanied by approximately twice that percentage reduction in the staining density. The ability to stain was lost when cartilage possessed approximately 42%, or less, of its normal hexuronic acid content.     

Synthesis of 24-nor-5 beta-cholan-23-oic acid derivatives: a convenient and efficient one-carbon degradation of the side chain of natural bile acids

An efficient procedure for obtaining nor-bile acids from natural (C24) bile acids is described. Treatment of formylated bile acids with sodium nitrite in a mixture of trifluoroacetic anhydride with trifluoroacetic acid gives, through a "second order" Beckmann rearrangement, 24-nor-23-nitriles. These compounds, on alkaline hydrolysis, afford the corresponding nor-bile acids in high yields. The sequence was successfully applied to the synthesis of 3 alpha-hydroxy-24-nor-5 beta-cholan-23-oic (norlithocholic) acid, 3 alpha,6 alpha- (norhyodeoxycholic), 3 alpha,7 alpha- (norchenodeoxycholic), 3 alpha,7 beta- (norursodeoxycholic), and 3 alpha,12 alpha-dihydroxy-24-nor-5 beta-cholan-23-oic (nordeoxycholic) acids, as well as 3 alpha,7 alpha,12 alpha-trihydroxy-24-nor-5 beta-cholan-23-oic (norcholic) acid. 13C-NMR spectra of their methyl esters are reported. The procedure provides a more rapid alternative to the Barbier-Wieland degradation for shortening by one methylene group the side chain of natural (C24) bile acids.