High-performance liquid chromatographic separation of ascorbic acid,erythorbic acid,dehydroascorbic acid,dehydroerythorbic acid,diketogulonic acid,and diketogluconic acid

High-performance liquid chromatography on a Zorbax NH₂ analytical column, with acetonitrile: 0.05 m KH₂PO₄ (75:25, $\text{w}\text{w}$) used as eluant, has allowed the separation, in less than 14 min, of ascorbic acid, erythorbic acid, dehydroascorbic acid, dehydroerythorbic acid, diketogulonic acid, and diketogluconic acid. Ultraviolet monitoring at 268 nm allows ascorbic acid and erythorbic acid to be detected at the 25-ng level, while refractive index detection monitors the elution of all six compounds. Tyrosine is a good internal standard, being well separated from the other compounds and having an adequate ultraviolet absorption at 268 nm. We have found dithiothreitol to be effective in rapidly reducing dehydroascorbic acid to ascorbic acid, providing the basis for indirectly determining dehydroascorbic acid after its reduction. The potential of this high-performance liquid chromatographic procedure for evaluating the levels of these compounds in orange juice and urine is demonstrated.     

Electrokinetic energy conversion by aqueous oxalic acid, citric acid, ascorbic acid, hippuric acid, and acetyl salicylic acid across urinary bladder membranes

Efficiency of energy conversion for electro-osmosis and streaming potential and the degree of coupling of acids across urinary bladder membranes of goat have been computed using non-equilibrium thermodynamic theory. The energy conversion maxima and degree of coupling for acids responsible for the formation of urinary calculi are found to be much low as compared to urea and urine.     

Quinolinic acid, alpha-picolinic acid, fusaric acid, and 2,6-pyridinedicarboxylic acid enhance the Fenton reaction in phosphate buffer.

Quinolinic acid, alpha-picolinic acid, fusaric acid, and 2,6-pyridinedicarboxylic acid enhanced the Fenton reaction in phosphate buffer, respectively. The enhancement by quinolinic acid, alpha-picolinic acid, fusaric acid, and 2,6-pyridinedicarboxylic acid of the Fenton reaction may be partly related to their respective actions in the biological systems such as a neurotoxic effect (quinolinic acid), a marked growth-inhibitory action on rice seeding (alpha-picolinic acid and fusaric acid), and an antiseptic (2,6-pyridinedicarboxylic acid). The ultraviolet-visible absorption spectrum of the mixture of alpha-picolinic acid with ferrous ion showed a characteristic visible absorbance band with a lambda(max) at 443 nm, suggesting that alpha-picolinic acid chelate of Fe2+ ion forms in the solution. Similar characteristic visible absorbance band was also observed for the mixture of Fe2+ ion with quinolinic acid (or fusaric acid, or 2,6-pyridinedicarboxylic acid). The chelation seems to be related to the enhancement by quinolinic acid, alpha-picolinic acid, fusaric acid, and 2,6-pyridinedicarboxylic acid of the Fenton reaction. alpha-Picolinic acid was reported to be a toxic substance isolated from the culture liquids of blast mould (Piricularia oryzae CAVARA). On the other hand, it has also been known that chlorogenic acid protects rice plants from the blast disease. The chlorogenic acid inhibited the formation of the hydroxyl radical in the reaction mixture of alpha-picolinic acid, FeSO4(NH4)2SO4, and H2O2. Thus the inhibition may be a possible mechanism of the protective action of the chlorogenic acid against the blast disease.     

Microbial production of chenodeoxycholic acid precursor, 12-ketochenodeoxycholic acid,from dehydrocholic acid

Summary Two kinds of bacteria (DC33 and DC1115) were isolated from soil as biotransformers of dehydrocholic acid to 12-ketochenodeoxycholic acid, and identified to be Brevibacterium fuscum and Lactobacillus xylosus, respectively. Dehydrocholic acid was converted via 7,12-diketolithocholic acid to 12-ketochenodeoxycholic acid by both strains, and the product and the intermediate were isolated and chemically identified. By using a jar fermentor, 12-ketochenodeoxycholic acid was produced with a more than 50% yield after 52 h by Brevibacterium fuscum with aerobic growth and anaerobic conversion, and after 24 h by Lactobacillus xylosus under anaerobic conditions, respectively.     

The preparation of bile acid amides and oxazolines, II, the synthesis of the amides and oxazolines of ursodeoxycholic acid, deoxycholic acid, hyodeoxycholic acid and cholic acid

Bile acid amides and oxazolines were synthesized by a sequence of steps involving the reaction of the free bile acid with formic acid to yield the formyloxy derivative, preparation of the formyloxy acid chloride, condensation of the acid chloride with 2-amino-2-methyl-1-propanol to give the amide and, finally, cyclization of the amide with thionyl chloride to give the oxazoline. The oxazolines were characterized by physical constants, thin layer and gas-liquid chromatography and identified by elemental analysis and gas-liquid chromatography-mass spectrometry. Some of the bile acid oxazoline derivatives alter the activity of bacterial 7-dehydroxylases $\text{in vitro}$, and inhibit the growth of certain anaerobic bacteria in pure culture.     

Fluorescent 2-aza-epsilon-adenosine derivatives of polyadenylic acid, polyuridylic acid, and polyinosinic acid.

A new fluorescent analog of adenosine, 1,N⁶-etheno-2-aza-adenosine, has been incorporated into polynucleotides by polynucleotide phosphorylase polymerization of 1,N⁶-etheno-2-aza-adenosine-5′-diphosphate and adenosine-5′-diphosphate, uridine-5′-diphosphate, or inosine-5′-diphosphate. These new oligonucletides possess high fluorescence when excited at 358 nm and emit at 495 nm. The ratio of the fluorescent and nonfluorescent portions of the copolymer can be controlled by the initial composition of the 2-aza-ε-adenosine-diphosphate and the corresponding nucleoside diphosphate. Fluorescent copolymers with a ratio varying from 1.6 to 35 have thus been synthesized. The physicochemical study of copolymers containing less than 10% of the 1,N⁶-etheno-2-aza-adenosine moiety showed that they are similar to poly(A), poly(U), or poly(I). Therefore, fluorescence and polarization study of the 1,N⁶-etheno-2-aza-adenosine residues that have been incorporated into the copolymer provides a sensitive indicator for the structure of the copolymer. Potentially these new copolymers may provide unique roles in probing the structure of poly(C) and poly(A) in cellular mRNA.     

Lipid metabolism in the perfused chicken liver. The uptake and metabolism of oleic acid, elaidic acid, cis-vaccenic acid, trans-vaccenic acid and stearic acid

Comparative studies were made of the uptake and metabolism of cis- and trans-octadecenoic acids by the perfused chicken liver. No differences were observed in the rates of uptake of the isomers. There was considerable incorporation of radioactivity into triglycerides and phospholipids, and some release of labelled lipid into the perfusate was observed. The cis-fatty acids were more readily incorporated into triglycerides than phospholipids, the reverse being true of the trans-fatty acids. Examination of the intramolecular distribution of fatty acids in triglycerides showed that the trans-fatty acid and stearate mainly occupied the 1- and 3-positions, and cis-fatty acids the 2-position. In the phospholipids phosphatidylcholine and phosphatidylethanolamine the trans-fatty acids again behaved like stearic acid and favoured the 1-position. No evidence was obtained of atypical patterns of uptake or metabolism of the trans-fatty acids.     

The non-oxidative decarboxylation ofp-hydroxybenzoic acid,gentisic acid,protocatechuic acid and gallic acid byKlebsiella aerogenes (Aerobacter aerogenes)

Klebsiella aerogenes adapted to a chemically-defined mineral salts medium with glucose orp-hydroxybenzoate as sole source of carbon and energy possessed constitutive decarboxylases for gentisate (2,5-dihydroxybenzoate), protocatechuate (3,4-dihydroxybenzoate) and gallate (3,4,5-trihydroxybenzoate) whose pH optima were respectively 5.9, 5.6 and 5.8. A decarboxylase for PHB was induced by PHB in both growing and resting cells; the induction was delayed or inhibited by chloramphenicol and by ultrasonic disruption of the bacteria. Crude ultrasonic preparations of PHB decarboxylase had an optimum pH of 6.0, a Michaelis constant of 4mm and an activation energy of 25,500 cal mole^–1 at 28 – 38 C. All four decarboxylations proceeded without O₂ and for every mole of phenolic acid decomposed one mole of CO₂ and one mole of the corresponding phenol were produced. The effects of ultrasonic disruption of the bacteria suggested that permeability barriers limited the rate of decarboxylation of PHB and 2,5-DHB but not of 3,4-DHB or 3,4,5-THB. During ultrasonic disintegration PHB and 3,4-DHB decarboxylases were retained solely by insoluble centrifugeable particles, whereas 2,5-DHB and 3,4,5-THB decarboxylases were gradually released into solution.The decarboxylation of protocatechuic acid is an essential stage in the assimilation ofp-hydroxybenzoic acid byK. aerogenes, whereas the decarboxylation ofp-hydroxybenzoate itself is an injurious side reaction.We wish to thank Mr. P. J. Wragg for technical assistance.     

Incorporation and distribution of dihomo-gamma-linolenic acid, arachidonic acid, and eicosapentaenoic acid in cultured human keratinocytes

Human keratinocytes in culture were labelled with 14C-dihomo-gamma-linolenic acid, 14C-arachidonic acid or 14C-eicosapentaenoic acid. All three eicosanoid precursor fatty acids were effectively incorporated into the cells. In phospholipids most of the radioactivity was recovered, in neutral lipids a substantial amount, and as free unesterified fatty acids only a minor amount. The most of the radioactivity was found in phosphatidylethanolamine which was also the major phospholipid as measured by phosphorous assay. The incorporation of dihomo-gamma-linolenic acid and arachidonic acid into lipid subfractions was essentially similar. Eicosapentaenoic acid was, however, much less effectively incorporated into phosphatidylinositol + phosphatidylserine and, correspondingly, more effectively into triacylglycerols as compared to the two other precursor fatty acids. Once incorporated, the distribution of all three precursor fatty acids was relatively stable, and only minor amounts of fatty acids were released into the culture medium during short term culture (two days). Our study demonstrates that eicosanoid precursor fatty acids are avidly taken up by human keratinocytes and esterified into membrane lipids. The clinical implication of this finding is that dietary manipulations might be employed to cause changes in the fatty acid composition of keratinocytes.     

Solid-liquid phase behavior of binary fatty acid mixtures. 2. Mixtures of oleic acid with lauric acid, myristic acid, and palmitic acid

Solid-liquid phase behavior was investigated for binary fatty acid mixtures composed of oleic acid (OA; cis-9-octadecenoic acid) and saturated fatty acids, lauric acid (LA; dodecanoic acid), myristic acid (MA; tetradecanoic acid), and palmitic acid (PA; hexadecanoic acid), by means of differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR). When the mixture was heated immediately after the solidification from the melt, the heat effect due to the gamma-to-alpha transformation of OA varied depending on the composition of the mixture. However, the mixture subjected to an annealing at the temperature slightly below the melting temperature provided the transformation at constant temperature which corresponds to the gamma-to-alpha transformation temperature of pure OA. This suggests that a solid phase formed by cooling of the melt of the mixture is not in an equilibrium state, but it relaxes to a stable solid during the annealing process. The T-X phase diagrams of these mixtures constructed from the DSC measurements demonstrate that the two fatty acid species are completely immiscible in a solid phase regardless of the type of polymorphs of OA, alpha- or gamma-form. According to a thermodynamic analysis of liquidus line basing on the regular solution model for the melt, the non-ideality of mixing tends to increase with the decrease in the acyl chain length of the saturated fatty acid, although the mixing is rather close to ideal.     

Dihomogammalinolenic acid, but not eicosapentaenoic acid, activates washed human platelets

Dihomogammalinolenic acid (2.5-20 microM) added to suspensions of washed human platelets induces platelet shape change and the formation of 1,2-diacylglycerol and phosphatidic acid, indicating the activation of phospholipase C. It also stimulates the phosphorylation of a 40 kDa protein, indicating the activation of protein kinase C. Dihomogammalinolenic acid is converted mainly to 12-hydroxyheptadecadienoic acid and to a smaller extent to prostaglandin E1 and thromboxane B1. Small quantities of the lipoxygenase product 12-hydroxyeicosatrienoic acid are also observed. Indomethacin, by blocking platelet cyclooxygenase, prevents the activation of phospholipase C, protein kinase C, and platelet shape change induced by dihomogammalinolenic acid. Compound UK 38485, a specific thromboxane synthetase inhibitor, does not block platelet activation induced by dihomogammalinolenic acid. The results indicate that endoperoxides derived from dihomogammalinolenic acid, such as prostaglandin G1 or prostaglandin H1, may be responsible for the stimulation of phospholipase C and protein kinase C, and for the induction of platelet shape change. Eicosapentaenoic acid does not activate platelets and is poorly metabolized by platelet cyclooxygenase and lipoxygenase. Eicosapentaenoic acid is a better inhibitor of platelet activation induced by various agonists in washed platelets than dihomogammalinolenic acid. Eicosapentaenoic acid and dihomogammalinolenic acid are, however, equally effective in inhibiting aggregation induced by collagen in platelet-rich plasma. We suggest that eicosapentaenoic acid might be a better antithrombotic agent than dihomogammalinolenic acid.     

In rat hepatocytes, myristic acid occurs through lipogenesis, palmitic acid shortening and lauric acid elongation

The origin of myristic acid in mammalian cells and the regulation of its endogenous cellular low concentration are not known. Another intriguing question is the potential metabolic properties of endogenous myristic acid as compared with exogenous myristic acid. In the present paper, we hypothesised and demonstrated that, in liver cells, in addition to the usual fatty acid synthase (FAS) pathway that produces predominantly palmitic acid and minor amounts of myristic acid, part of endogenous cellular myristic acid also comes from a shortening of palmitic acid, likely by peroxisomal β-oxidation and from lauric acid by elongation. From a nutritional point of view, C16:0 is universally found in natural fats and its shortening to myristic acid could contribute to a non-negligible source of this fatty acid (FA) in the organism. Then, we measured the distribution of endogenously synthesised myristic acid in lipid species and compared it with that of exogenous myristic acid. Our results do not support the hypothesis of different metabolic fates of endogenous and exogenous myristic acid and suggest that whatever the origin of myristic acid, its cellular concentration and lipid distribution are highly regulated.     

The kinetics and mechanisms of reactions of iron(III) with caffeic acid, chlorogenic acid, sinapic acid, ferulic acid and naringin

The kinetics and mechanisms of the reactions of iron(III) with the hydroxy cinnamic acid based ligands caffeic, chlorogenic, sinapic and ferulic acids and the flavonoid naringin have been investigated in aqueous solution. The mechanisms for caffeic and chlorogenic acid are generally consistent with the formation of a 1:1 complex that subsequently decays through an electron transfer reaction. On reaction with iron(III), ferulic and sinapic acids undergo an electron transfer without the prior formation of any complex. There was no evidence of electron transfer occurring in the complex formed when iron(III) is reacted with naringin. Rate constants for k1 (formation) and k(-1) (dissociation) have been evaluated for the complex formation reactions of [Fe(H2O)6(OH)]2+ with caffeic acid, chlorogenic acid and naringin. Analysis of the kinetic data yielded stability constants, equilibrium constants for protonation of the iron(III) chlorogenic acid complex initially formed, together with the rate constants for complex decomposition through intramolecular electron transfers and in the case of caffeic acid and chlorogenic acid, rate constants for the iron(III) assisted decomposition of the initial complex formed. Some of the suggested mechanisms and calculated rate constants are validated by calculations carried out using global analysis of time dependent spectra.     

Sempervirenic acid,a diterpene acid from Solidago sempervirens

Sempervirenic acid, a new diterpene has been isolated from Solidago sempervirens and its structure determined by spectroscopic methods and chemical conversions to be 3β-acetoxy-labda-7,13-diene-15-oic acid.