Hanne H. F. Refsgaard

Oxidation of bovine serum albumin initiated by the Fenton reaction—effect of EDTA, tert-butylhydroperoxide and tetrahydrofuran

Oxidation of bovine serum albumin (BSA) was investigated using different oxidants: The water-soluble azo-initiator 2,2′azo-bis-(2-amidinopropane) hydrochloride (AAPH), a combination of FeCl3 and ascorbate or the Fenton oxidant consisting of FeCl2, H2O2 and EDTA. In addition, the effects of exogenous compounds such as tert-butyl hydroperoxide (tBuOOH) or solvents such as tetrahydrofuran (THF), often used in model systems, was evaluated. The extent of protein damage was studied by measuring protein carbonyl groups and protein hydroperoxides. The interaction between Fenton oxidant and EDTA, THF or tBuOOH was further characterized using spin trapping electron spin resonance (ESR) spectroscopy. The results showed that the extent of protein oxidation depended on the oxidant used. The Fenton oxidant was the most reactive of the initiators tested. However, in the absence of EDTA, the Fenton system produced protein carbonyl groups on BSA equivalent to that obtained with the other oxidants, however, significantly more protein hydroperoxide was produced. Surprisingly, it was also found that addition of tBuOOH or THF to BSA reduced protein damage when the oxidation was initiated with the Fenton oxidant. ESR investigation showed that EDTA played a key role in the generation of free radicals. It was also revealed that in an EDTA containing system both tBuOOH and THF were able to react with radicals without inducing protein damage in effect protecting BSA from oxidative damage.

Increase of neuronal histamine in obese rats is associated with decreases in body weight and plasma triglycerides.

Objective: The purpose of the present study was to examine the metabolic effects of a specific histamine H3 receptor antagonist, the cinnamic amide NNC 0038‐0000‐1202 (NNC 38‐1202).

Evaluation of activity of selected antioxidants on proteins in solution and in emulsions.

Protection against protein oxidation by lipophilic and hydrophilic antioxidants in model systems using bovine serum albumin (BSA) in solution alone, or in an emulsion with linolenic acid methyl ester (LnMe) was found to be strongly dependent on the oxidation initiator. Tocopherol, Trolox, or the carotenoids astaxanthin and canthaxanthin were incubated with BSA or BSA/LnMe and oxidation was initiated either with the water-soluble azo-initiator 2,2′ azo-bis-(2-amidinopropane) hydrochloride (AAPH), or FeCl3 and ascorbate, or the Fenton system using FeCl2/EDTA/H2O2, or with the singlet oxygen generating species anthracene-9,10-dipropionic acid disodium 1,4 endoperoxide (NDPO2). The results show that all the antioxidants tested were inefficient in the system with FeCl3/ascorbate. However, with the other initiating agents, the hydrophilic antioxidant, Trolox, was the most effective in preventing both protein and lipid oxidation. In contrast the lipophilic antioxidants were ineffective in preventing oxidation of BSA in aqueous solution, but did show some moderate antioxidative activity on protein and lipid in the BSA/LnMe system. Using the singlet oxygen generating system it was also demonstrated that Trolox always provided better protection of the protein than tocopherol and the carotenoids in both the BSA and the BSA/LnMe systems. In conclusion, prevention of protein oxidation using a water-soluble antioxidant has a protective effect on the lipid fraction and this approach deserves further attention in complex biological systems.

Light sensitivity of colourants used in alcoholic beverages

ZusammenfassungUm einen objektiven Wert für die Empfindlichkeit von Farbstoffen in alkoholischen Getränken gegenüber ultraviolettem und sichtbarem Licht festlegen zu können, wurde die Quantenausbeute für Photobleichung in ethanolischen Lösungen von Hexacol Red 2G Supra (E 128), Hexacol Sunset Yellow FCF Supra (E110), Caramel Farbe (E 150) und Liquid Red Colour (E 128; auf Carminsäure basieren) bei 25 °C in kontinuierlichen Photolyse-Experimenten mit monochromatischem Licht mit den Wellenlängen 334 nm, 366 nm und 436 nm bestimmt. Die Quantenausbeute für Photobleichung von Carminsäure in Liquid Red Colour in Fruchtwein (pH 3,1 mit 15%, v/v, Ethanol) war 2,1·10−2 Mol·Einstein−1 bei 334 nm; 1,3·10−3 Mol·Einstein−1 bei 366 nm und 5,6·10−5 Mol·Einstein−1 bei 436 nm. Die Quantenausbeute für Hexacol Sunset Yellow FCF Supra in Fruchtwein war 5,9·10−4 Mol·Einstein−1 bei 334 nm und 2,4·10−4 Mol·Einstein−1 bei 366 nm. Die Quantenausbeute für Photobleichung für Hexacol Red 2G Supra war 3,7·10−5 Mol·Einstein−1 bei 366 nm Bestrahlung. Für Hexacol Red 2G Supra und Hexacol Sunset Yellow FCF Supra war die Quantenausbeute bei 436 nm Bestrahlung geringer als 10−6 Mol·Einstein−1, das gleiche gilt für Hexacol Red 2G Supra bei 334 nm Bestrahlung. Folgende Reihenfolge der Lichtempfindlichkeit in Fruchtwein wurde festgestellt (bei 366 nm): Liquid Red Colour > Hexacol Sunset Yellow FCF Supra > Hexacol Red 2G Supra. Caramel Farbe in Aquavit (pH 7,1, mit 42%, v/v, Ethanol) war mäßig lichtempfindlich; die Quantenausbeute war fast unabhängig von der Bestrahlungswellenlänge. Relative Quantenausbeuten von 1,6 bei 334 nm, 1,0 bei 366 nm und 1,0 bei 436 nm wurden bestimmt. Der letzte Wert entspricht 2,6 g Caramel per Einstein.AbstractQuantum yields for photobleaching in ethanol solutions of Hexacol Red 2G Supra (E 128), Hexacol Sunset Yellow FCF Supra (E 110), caramel colour (E 150) and Liquid Red Colour (E 128) based on carminic acid, have been determined at 25° C in continuous photolysis experiments using monochromatic light at each of three wavelengths 334 nm, 366 nm and 436 nm, in order to obtain an objektive measure of the sensitivity to ultraviolet and visible light of colourants used in alcoholic beverages. For photobleaching of carminic acid from Liquid Red Colour in fruit wine [pH 3.1, with 15% (v/v) ethanol] quantum yield was determined as 2.1·10−2 mol·einstein−1 for 334 nm irradiation, 1.3·10−3 mol·einstein−1 for 366 nm irradiation and 5.6·10−5 mol·einstein−1 for 436 nm irradiation, respectively. For Hexacol Sunset Yellow FCF Supra in fruit wine, the quantum yield for 334 nm irradiation was determined as 5.9·10−4 mol·einstein−1 and for 366 nm irradiation as 2.4·10−4 mol·einstein−1. Hexacol Red 2G Supra in fruit wine had a quantum yield of 3.7·10−5 mol·einstein−1 for 366 nm irradiation. For Hexacol Red 2G Supra and Hexacol Sunset Yellow FCF Supra for 436 nm irradiation and for Hexacol Red 2G Supra for 334 nm irradiation the quantum yields were less than 10−6 mol·einstein−1. The following order was determined for light sensitivity in fruit wine (366 nm light): Liquid Red Colour > Hexacol Sunset Yellow FCF Supra > Hexacol Red 2G Supra. The caramel colour in aquavit [pH 7.1, with 42% (v/v) ethanol] was moderately sensitive to light with a quantum yield almost independent of irradiation wavelength. Relative quantum yields were determined as 1.6 for 334 nm irradiation, 1.0 for 366 nm and 1.0 for 436 nm irradiation. The latter value corresponded to 2.6 g caramel per einstein absorbed.

In silico modelling of permeation enhancement potency in Caco-2 monolayers based on molecular descriptors and random forest

Structural traits of permeation enhancers are important determinants of their capacity to promote enhanced drug absorption. Therefore, in order to obtain a better understanding of structure-activity relationships for permeation enhancers, a Quantitative Structural Activity Relationship (QSAR) model has been developed. The random forest-QSAR model was based upon Caco-2 data for 41 surfactant-like permeation enhancers from Whitehead et al. (2008) and molecular descriptors calculated from their structure. The QSAR model was validated by two test-sets: (i) an eleven compound experimental set with Caco-2 data and (ii) nine compounds with Caco-2 data from literature. Feature contributions, a recent developed diagnostic tool, was applied to elucidate the contribution of individual molecular descriptors to the predicted potency. Feature contributions provided easy interpretable suggestions of important structural properties for potent permeation enhancers such as segregation of hydrophilic and lipophilic domains. Focusing on surfactant-like properties, it is possible to model the potency of the complex pharmaceutical excipients, permeation enhancers. For the first time, a QSAR model has been developed for permeation enhancement. The model is a valuable in silico approach for both screening of new permeation enhancers and physicochemical optimisation of surfactant enhancer systems.

Solid-state13C NMR investigations of insoluble deposits in aromatic bitters

Insoluble deposit formed in aromatic bitters produced from alcoholic extracts of about 30 different botanical materials with a dry matter content of 1.67% and 38 vol% alcohol and a characteristic astringent flavour was shown by13C NMR spectroscopy to be a coprecipitate of plant polyphenols, proteins and carbohydrates. The polyphenols in the deposit were flavonoids such as anthocyanidins and anthocyanins, with glucose as the principal carbohydrate and derivatives of cinnamic acid. The deposit formation in the aromatic bitters was accelerated by addition of the radical initiator 2,2′-azobis(2-amidinopropane) dihydrochloride or by addition of hydrogen peroxide in combination with iron(III) nitrate and ascorbic acid as a Fenton reagent. Polymerization of catechin and pelargonidin was shown, in model experiments, to be oxidative and to be accelerated by exposure to light. A mechanism involving oxidative polymerization of polyphenols followed by coprecipitation of polymerized polyphenols, proteins and carbohydrates is proposed for formation of deposits in alcoholic beverages of the bitters type.

Light-induced sensory and chemical changes in aquavit

Aquavit in clear bottles was stored in sunlight, in fluorescent light and in the dark under conditions relevant for the retail trade. Every 2 wk over a 6 mo period, the odour and taste of the aquavit were characterized by descriptive sensory analysis and concentrations of volatile compounds were determined together with tristimulus colour analysis. Formation of geranium-leaf odour and taste were the most pronounced light-induced sensory changes. The colour of the samples stored in light was determined less green and yellow and the samples were bleached. Concentrations of many of the volatile compounds characteristic of aquavit, including carvone, decreased in light. Carvonecamphor and limonene photoproducts were formed by photochemical reactions of carvone and limonene, respectively. None of these photoproducts had geranium-leaf odour by gas chromatography-olfactometry, but a compound with low odour threshold had. The compound could not be detected by flame ionization. A photochemical reactor suitable for accelerated light storage of beverages was constructed. The sensory and chemical changes found after 8h irradiation in the reactor were similar to those taking place in aquavit stored for 4 mo in sunlight.

Spatial distribution of soluble insulin in pig subcutaneous tissue: Effect of needle length, injection speed and injected volume

The spatial distribution of a soluble insulin formulation was visualized and quantified in 3-dimensions using X-ray computed tomography. The drug distribution was visualized for ex vivo injections in pig subcutaneous tissue. Pig subcutaneous tissue has very distinct layers, which could be separated in the tomographic reconstructions and the amount of drug in each tissue class was quantified. With a scan time of about 45min per sample, and a robust segmentation it was possible to analyze differences in the spatial drug distribution between several similar injections. It was studied how the drug distribution was effected by needle length, injection speed and injected volume. For an injected volume of 0.1ml and injection depth of 8mm about 50% of the injections were partly intramuscular. Using a 5mm needle resulted in purely subcutaneous injections with minor differences in the spatial drug distribution between injections. Increasing the injected volume from 0.1ml to 1ml did not increase the intramuscular volume fraction, but gave a significantly higher volume fraction placed in the fascia separating the deep and superficial subcutaneous fat layers. Varying the injection speed from 25l/s up to 300l/s gave no changes in the drug concentration distribution. The method presented gives novel insight into subcutaneous injections of soluble insulin drugs and can be used to optimize the injection technique for subcutaneous drug administration in preclinical studies of rodents.

Light-induced sensory and chemical changes in aromatic bitters

Every month for a year, samples of aromatic bitters stored in clear and green bottles under conditions relevant to the retail trade, (1) in sunlight, (2) in fluorescent light and (3) in the dark, were characterized by descriptive sensory analysis, tristimulus colorimetry and by the concentrations of 13 flavour compounds. Aromatic bitters stored in light developed a higher intensity of sweet and solvent-like odour, described by the assessors as catty-like or ribes. Bitters stored in light had a stronger tendency to become turbid and these samples had a fortified-wine-like flavour and were less bitter and less astringent. A new method in this context, growth curve analysis, was used successfully to detect significant time and treatment effects in the sensory data. By partial least-squares analysis only a minor part of the light-induced sensory effects in bitters could be accounted for by the concentration of flavour compounds.