Isolation and identification of oxidation products of syringol from brines and heated meat matrix.

In this study we developed new extraction and detection methods (using HPLC-UV and LC-MS), making it possible to analyze the smoke phenol syringol and its oxidation products nitrososyringol, nitrosyringol, and the syringol dimer 3,3',5,5'-tetramethoxy-1,1'-biphenyl-4,4'-diol, which were identified in heated meat for the first time. Preliminary brine experiments performed with different concentrations of ascorbic acid showed that high amounts of this antioxidant also resulted in almost complete degradation of syringol and to formation of the oxidation products when the brines were heated at low pH values. Heat treatment (80°C) and subsequent simulated digestion applied to meat samples containing syringol, ascorbic acid and different concentrations of sodium nitrite produced 3,3',5,5'-tetramethoxy-1,1'-biphenyl-4,4'-diol even at a low nitrite level in the meat matrix, while nitroso- and nitrosyringol were isolated only after the digestion experiments. Increasing amounts of oxygen in the meat matrix decreased the syringol concentration and enhanced the formation of the reaction products in comparison to the samples without added oxygen.

Isolation and identification of oxidation products of guaiacol from brines and heated meat matrix.

In this study we investigated the formation of the oxidation products of guaiacol in brines and heated meat matrix: 6-nitrosoguaiacol, 4-nitroguaiacol and 6-nitroguaiacol. For this purpose we applied a newly developed HPLC-UV and LC-MS method. For the first time, 6-nitrosoguaiacol was determined in brine and meat (containing guaiacol and sodium nitrite), which had been heated to 80°C and subsequently subjected to simulated digestion. Application of 500mg/L ascorbic acid to the brines reduced guaiacol degradation at pH3 and simultaneously inhibited the formation of 6-nitrosoguaiacol compared to brines containing only 100mg/L of ASC. The oxidation products were isolated with a new extraction method from meat samples containing 400mg/kg sodium nitrite at pH3.6 following simulated digestion. When oxygen was added, 6-nitrosoguaiacol was determined even at legally allowed levels (150mg/kg) of the curing agent. Finally, we developed a new LC-MS method for the separation and qualitative determination of the four main smoke methoxyphenols.

HPLC Separation of Vitamin E and Its Oxidation Products and Effects of Oxidized Tocotrienols on the Viability of MCF-7 Breast Cancer Cells in Vitro.

Tocotrienols, a vitamin E subgroup, exert potent anticancer effects, but easily degrade due to oxidation. Eight vitamin E reference compounds, α-, ß-, γ-, or δ-tocopherols or -tocotrienols, were thermally oxidized in n-hexane. The corresponding predominantly dimeric oxidation products were separated from the parent compounds by diol-modified normal-phase HPLC-UV and characterized by mass spectroscopy. The composition of test compounds, that is, α-tocotrienol, γ-tocotrienol, or palm tocotrienol-rich fraction (TRF), before and after thermal oxidation was determined by HPLC-DAD, and MCF-7 cells were treated with both nonoxidized and oxidized test compounds for 72 h. Whereas all nonoxidized test compounds (0-100 µM) led to dose-dependent decreases in cell viability, equimolar oxidized α-tocotrienol had a weaker effect, and oxidized TRF had no such effect. However, the IC50 value of oxidized γ-tocotrienol was lower (85 µM) than that of nonoxidized γ-tocotrienol (134 µM), thereby suggesting that γ-tocotrienol oxidation products are able to reduce tumor cell viability in vitro.

Changes in the protein secondary structure of hen's egg yolk determined by Fourier transform infrared spectroscopy during the first eight days of incubation.

In this study, incubation-induced alterations in the protein secondary structures of egg yolk and its major fractions (granules, plasma, and low-density lipoproteins [LDL]) were monitored during the first 8 d of embryogenesis using Fourier transform infrared spectroscopy (FTIR) and isoelectric focusing (IEF). Two factors potentially connected with egg yolk protein secondary structure changes were evaluated, i.e., the pH value of incubated egg yolk, and phosvitin, an important egg yolk protein assumed to play an important role in hematopoiesis as the iron carrier during early embryogenesis. However, neither the significant increase in pH value (6.07 to 6.92) of egg yolk during incubation of fertilized eggs, nor the release of iron from phosvitin were found to be directly related to the changes in protein secondary structure in egg yolk and its fractions. FTIR showed that the protein conformation in whole egg yolk, granules, and LDL was stable during incubation, but separate evaluation of the plasma fraction revealed considerable changes in secondary structure. However, it is unlikely that these changes were provoked by structure changes of the proteins originally present in plasma; instead, the physiological influx of albumen into the yolk sac was expected to play an important role in the protein modifications of egg yolk, as was shown both by FTIR and IEF of the water-soluble egg yolk proteins. Moreover, FTIR was used to determine the naturally occurring proportions (%) of the secondary structure elements in egg yolk and its 3 fractions on d 0 of incubation. The granules fraction mainly consisted of a mixture of inter- and intramolecular ß-sheets (57.04%±0.39%). The plasma fraction was found to consist mainly of α-helices (43.23%±0.27%), whereas LDL was composed almost exclusively of intramolecular ß-sheets (67.36%±0.56%) or ß-turns, or both. On the other hand, whole egg yolk was mainly composed of intermolecular ß-sheets (39.77%±0.48%), potentially indicating molecular interchanges between the individual fractions.

Determination of heat-induced changes in the protein secondary structure of reconstituted livetins (water-soluble proteins from hen's egg yolk) by FTIR.

This study characterized the impact of technological treatments on the protein secondary structure of a newly developed egg yolk livetin formulation and its components α-livetin, which is identical with chicken serum albumin, and γ-livetin, the bioactive antibody immunoglobulin Y. Fourier transform infrared (FTIR) spectroscopy at 25 °C revealed that the largest proportion of conformal elements comprised intramolecular (native) ß-sheets (60-80%) in γ-livetin, and α-helices/random coils (60.59%) in α-livetin. In reconstituted freeze-dried livetins, the main protein conformations were also intramolecular (native) ß-sheets (55.08%) and α-helices/random coils (30.51%), but upon heating from 25 to 95 °C, the former decreased sigmoidally at the onset-of-denaturation temperature (TOD (FTIR)) of 69.5 °C, concomitant with a sigmoidal increase in intermolecular (denatured) ß-sheets at a TOD (FTIR) of 72.4 °C and a sigmoidal decrease in IgY activity at TOD (ELISA) of 67.5 °C. Reconstituted spray-dried livetins showed less native ß-sheets and significantly lower TOD (FTIR) values than freeze-dried livetins.

Stability of α-tocotrienol and α-tocopherol in salami-type sausages and curing brine depending on nitrite and pH.

We studied the stability of the valuable vitamer nutrients α-tocotrienol and α-tocopherol and options for their protection in salami-type sausages (blended with α-tocotrienol-rich barley oil) and curing brine. Four different sausage formulations were produced containing nitrite curing salt; nitrite curing salt and ascorbic acid (300mg/kg); nitrite curing salt and carnosic acid (45mg/kg); or sodium chloride. Initial vitamer contents (100mg/kg) did not decrease significantly during ripening and decreased only slightly during storage. Ascorbic acid and carnosic acid were found to be effective in preserving the vitamers in fresh sausages. Freeze-drying of sausages resulted in a significant loss of vitamers (97%), particularly after 14-day storage at room temperature, even in the presence of shielding gases. The vitamer content in the curing brine decreased with decreasing pH in the presence of nitrite. A nitrite concentration of 136mg/L at pH4 resulted in significant loss (90%) of the vitamers. Sufficient stability of the vitamers in salami-type sausage and curing brine can be achieved by processing, formulation, and storage conditions.

Comparison of dietary tocotrienols from barley and palm oils in hen's egg yolk: transfer efficiency, influence of emulsification, and effect on egg cholesterol.

BACKGROUND: The main component in tocotrienols (T3) from barley (Hordeum vulgare L.) is α-T3, the vitamer with the highest bioavailability, while palm oil T3 is particularly rich in γ-T3. Unlike tocopherols, T3 are known for their cholesterogenesis-inhibiting, neuroprotective and anticarcinogenic properties. In this study the oral bioavailabilities of T3 from barley oil (3.98 mg day⁻¹) and T3 from palm oil (3.36 mg day⁻¹) in nanoemulsified formulations (NE) and self-emulsifying systems (SES) were compared using hen's eggs as a bioindicator. In addition, the transfer efficiencies of barley oil T3 and palm oil T3 into egg yolk were compared, as well as their effects on egg cholesterol levels. RESULTS: Nanoemulsification led to T3 levels (132.9 µg per egg) higher than with non-emulsified barley oil (112.8 µg per egg) and barley oil SES (116.7 µg per egg) owing to the high proportions of α-T3 (99-117 µg per egg), which has a particularly high transfer efficiency (4.32-6.75%). T3 contents of eggs from hens fed barley oil supplements (112-132 µg per egg) were significantly higher than those of eggs from hens fed palm oil supplements (70-78 µg per egg). Addition of barley and palm oils to laying hen feed decreased egg yolk cholesterol by 4 and 6% respectively. CONCLUSION: Results from this animal study may help to establish T3 from barley as a dietary supplement and to develop nutritionally improved hen's eggs.

Identification and characterization of dimeric oxidation products of p-cymene-2,3-diol isolated from Thymus vulgaris L.

The aim of this study was to investigate the oxidation products of p-cymene-2,3-diol, a major antioxidative constituent of thyme (Thymus vulgaris L.). Although a dimeric form of p-cymene-2,3-diol and some derivative substances exhibiting valuable food technological and health-promoting properties have been reported in earlier publications, no obvious correlation has been shown between these substances. A modified HPLC-ESI-MS method made it possible to prove that two dimers, 3,4,3',4'-tetrahydroxy-5,5'-diisopropyl-2,2'-dimethylbiphenyl (1) and the newly identified 3',4'-dihydroxy-5,5'-diisopropyl-2,2'-dimethylbiphenyl-3,4-dione (2), are oxidation products of p-cymene-2,3-diol. 2 was characterized by the fragmentation pattern determined by multiple mass spectrometry, (1)H NMR, (13)C NMR, H-H COSY, HSQC, and HMBC. Both biphenyls were also quantitated in freeze-dried thyme as well as in a food matrix spiked with thyme extract. Model experiments using raw and cooked minced pork meat as matrix and sodium nitrite as oxidizing and reduction agent with and without ascorbic acid as protective reagent showed the correlation between food processing and dimer generation.

Identification of α-tocotrienolquinone epoxides and development of an efficient molecular distillation procedure for quantitation of α-tocotrienol oxidation products in food matrices by high-performance liquid chromatography with diode array and fluorescence detection.

The aim of this study was to investigate the most important oxidation products of α-tocotrienol (α-T3) along with other tocochromanols in lipid matrices and tocotrienol-rich foods. For this purpose, an efficient molecular distillation procedure was developed for the extraction of analytes, and α-T3-spiked and thermally oxidized natural lipids (lard and wheat germ oil) and α-T3-rich foods (wholemeal rye bread and oil from dried brewer's spent grain) were investigated through HPLC-DAD-F. The following α-T3 oxidation products were extractable from lipid matrices along with tocochromanols: α-tocotrienolquinone (α-T3Q), α-tocotrienolquinone-4a,5-epoxide (α-T3Q-4a,5-E), α-tocotrienolquinone-7,8-epoxide (α-T3Q-7,8-E), 7-formyl-ß-tocotrienol (7-FßT3), and 5-formyl-γ-tocotrienol (5-FγT3). Recovery rates were as high as 88% and enrichment factors up to 124. The proposed method allows the investigation of α-T3Q, α-T3Q-4a,5-E, α-T3Q-7,8-E, 7-FßT3, and 5-FγT3 in small quantities (<0.78 µg/g) in lipid matrices, which is necessary for the investigation and analysis of the formation kinetics of these oxidation products in fat, oils, and tocotrienol-rich foods.

Comparison of 51chromium-labeled ethylenediamine tetra-acetic acid and iohexol as blood markers for intestinal permeability testing in Beagle dogs.

(51)Chromium-labeled ethylenediamine tetra-acetic acid ((51)Cr-EDTA) is the gold standard probe for assessing intestinal permeability (IP) in dogs, but exposure to radioactivity is a disadvantage. Iohexol is a safe contrast medium commonly used for medical imaging purposes and has been successfully applied more recently for the assessment of IP in animal models and humans. This study aimed at comparing (51)Cr-EDTA and iohexol as IP blood markers in dogs. A test solution containing (51)Cr-EDTA and iohexol was administered intragastrically to seven healthy laboratory Beagle dogs, and percentage recoveries in serum were calculated. The strong linear association (correlation, r=0.76 and linear regression, y=0.03+5.04x) between (51)Cr-EDTA and iohexol supports the potential usefulness of iohexol as an IP blood marker in dogs.

Separation of α-tocotrienol oxidation products and eight tocochromanols by HPLC with DAD and fluorescence detection and identification of unknown peaks by DAD, PBI-EIMS, FTIR, and NMR.

Tocotrienols, like tocopherols, are members of the vitamin E family. While tocopherols (T) have been studied intensively, only recently have tocotrienols (T3) received increased attention due to their special health benefits. However, these positive attributes of T3 are probably lost as a result of degradation during food storage and processing, and there is little information about their oxidation products. Of particular interest are the oxidation products of α-tocotrienol (α-T3) as this is the least thermostable T3 isomer with the highest rate of degradation. The objective of this study was therefore to develop a reliable method for the determination of the most important oxidation products of α-T3 along with other tocochromanol isomers. We developed a high-performance liquid chromatography method with diode array detection, fluorescence detection, and a particle beam interface electron impact mass spectroscopy in order to separate the most important oxidation products of α-T3 (α-T3 spirodimers/spirotrimers, α-tocotrienoldihydroxy dimer, 7-formyl-ß-tocotrienol (7-FßT3), 5-formyl-γ-tocotrienol (5-FγT3), α-tocotrienolquinone (α-T3Q), and α-T3Q dimers and α-tocotrienolquinone epoxides (α-T3QE)) from eight tocochromanol isomers. Furthermore, we sought to identify the as yet unknown oxidation products 5-FγT3, 7-FßT3, α-T3Q-dimer, and α-T3QE. Of these, 5-FγT3 was fully characterized by Fourier transform infrared spectroscopy and (1)H and (13)C nuclear magnetic resonance spectroscopy.

The phenolic acids from bacterial degradation of the mangiferin aglycone are quantified in the feces of pigs after oral ingestion of an extract of Cyclopia genistoides (honeybush tea).

Polyphenols are cleaved by bacterial enzymes to form phenolic acid metabolites in the colon, where they may exert physiologic effects. For norathyriol, the aglycone of mangiferin, one of the major phenolic compounds present in Cyclopia genistoides (honeybush), a further bacterial degradation is likely; but knowledge of the importance of this metabolic process is very limited. Based on a hypothesized cleavage of the middle ring of norathyriol, this study was designed to determine phenolic cleavage products in the feces of pigs fed an extract of C genistoides. Pigs received 74 mg mangiferin per kilogram of body weight daily for 11 days; feces fractions were collected on day 11 and on the first 2 days after the last intake of extract. Several phenolic acids were detected in the feces samples, including 3-hydroxyphenylacetic acid; 4-hydroxybenzoic acid; 3,4-dihydroxybenzoic acid; 3,4-dihydroxyphenylacetic acid; 2,4,6-trihydroxybenzoic acid; 3,4,5-trihydroxybenzoic acid (gallic acid); and phloroglucinol. However, in vivo formation was likely only for 3,4-dihydroxybenzoic acid; 3,4-dihydroxyphenylacetic acid; 2,4,6-trihydroxybenzoic acid; and 3,4,5-trihydroxybenzoic acid because these were not present in the blank feces, in the animals' normal diet, or in the C genistoides extract. The fact that these amounts were very low suggests further degradation of the metabolites by intestinal microflora or absorption of the cleavage products by the colon.

Detergent decellularization of heart valves for tissue engineering: toxicological effects of residual detergents on human endothelial cells.

Detergents are powerful agents for tissue decellularization. Despite this, the high toxicity of detergent residua can be a major limitation. This study evaluated the efficacy of detergent removal from decellularized pulmonary valves (PVs) and the consequences of repopulation with human endothelial cells (HECs). Porcine PVs were treated with 1% sodium deoxycholate (SDC), group A; 1% sodium dodecyl sulfate (SDS), group B; and a mixture of 0.5% SDC/0.5% SDS, group C (n = 5 each). After each of 10 succeeding wash cycles (WCs), samples of the washing solution (WS) were analyzed by solid phase extraction and high performance liquid chromatography for the presence of detergents. Metabolic activity of HEC was also assessed in the WS samples (cytotoxicity and MTS assays). Decellularized and washed PVs were reseeded with HEC. Histological analysis demonstrated efficient tissue decellularization in all groups. Detergents' concentration in all WSs decreased exponentially and was below 50 mg/L after 6, 8, and 4 WCs in groups A, B, and C, respectively. This concentration resulted in no significant toxic influence on cell cultures, and scaffolds could be efficiently reseeded with HEC. In conclusion, intensive washing of detergent decellularized valvular scaffolds lowers the residual contamination below a hazardous threshold and allows their successful repopulation with HEC for tissue engineering purposes.

Estimation of intestinal permeability in healthy dogs using the contrast medium iohexol.

BACKGROUND: Iohexol is a nonradioactive marker that has been used successfully to test intestinal permeability in humans with inflammatory bowel disease. There is evidence in dogs that iohexol shares a similar permeability pathway as (51)chromium-EDTA, the gold standard marker. OBJECTIVE: The objective of this study was to determine an optimal oral iohexol dosage for an intestinal permeability serum test (IPST) and to use the test to estimate intestinal permeability in healthy dogs. METHODS: Eight clinically healthy dogs free of gastrointestinal, liver, and pancreatic disease were used in the study. Dosages of 0.25, 0.5, 1.0, 2.0, and 4.0 mL/kg of Omnipaque-350 (iohexol) were administered to 2 dogs at weekly intervals. Iohexol concentration was determined in serum samples obtained hourly for 6 hours after administration by high-performance liquid chromatography. Using the optimal dosage, iohexol was administered to 8 dogs twice, 6-36 days (mean 10 days) apart, and coefficients of variation (CVs) for iohexol concentration were calculated. RESULTS: A dosage of 2.0 mL/kg was chosen as optimal for the IPST, based on ease of iohexol detection in serum, intestinal contrast, and clinical effects of iohexol. Following administration of this dose to healthy dogs, mean (+/-SD) serum iohexol concentrations were 8.74+/-4.38, 11.89+/-5.67, 12.40+/-5.47, 9.23+/-5.54, 7.61+/-5.13, and 5.27+/-2.67 microg/mL at 1, 2, 3, 4, 5, and 6 hours after iohexol administration, respectively. CVs between the 2 test days were 28-45%. CONCLUSIONS: Using the iohexol dosage established in this study, the iohexol IPST was easy to perform as a marker for intestinal permeability in dogs. Further studies to establish reference intervals and evaluate the diagnostic value of the iohexol IPST in dogs with gastrointestinal disease are warranted.

Aspalathin, a flavonoid in Aspalathus linearis (rooibos), is absorbed by pig intestine as a C-glycoside.

Aspalathin, a dihydrochalcone and C-glycoside, is the most abundant flavonoid in rooibos (Aspalathus linearis), which is well known as an herbal tea in many countries. Aspalathin appears to have in vitro antioxidative and antimutagenic effects. To understand the effects of aspalathin in the body, research on the absorption in the intestinal tract, metabolism in the body, and identification of circulating metabolites in vivo is required. We investigated the metabolism of aspalathin to identify the parent compound and related metabolites in urine and plasma after orally administering a rooibos extract (16.3% aspalathin by 96 g rooibos extract, which equates to 1.1 kg dried rooibos material), produced from unfermented rooibos plant material, to pigs over a period of 11 days (oral dosage, 157-167 mg aspalathin per kg body weight daily). On days 7 and 11 of the study and days 1 and 2 after termination, urine was collected in 24-hour fractions, and plasma samples were collected at various time points. To our knowledge, this is the first time aspalathin metabolites have been identified in vivo, by presenting evidence of the absorption of aspalathin. Six substances identified in the urine by liquid chromatography-mass spectrometry were identified; these represent aspalathin and the metabolites methylated aspalathin, glucuronidated aspalathin glucuronidated and methylated aspalathin, a glucuronidated aglycone of aspalathin, as well as a metabolite of eriodictyol. The latter compound was methylated and contained 2 glucuronic acid moieties. This study showed that aspalathin can be absorbed by the intestine as C-glycoside as well as being cleaved in an aglycone and sugar moiety. The major metabolite in the enzymatically treated samples was methylated aspalathin. Between 0.1% and 0.9% of the administered dose of aspalathin could be detected in the urine on days 7 and 11 of the feeding study. No metabolites or aspalathin were found in plasma samples. The identification of the metabolites in vivo enables investigations to determine the biological potential of rooibos extracts.

Mangiferin and hesperidin metabolites are absorbed from the gastrointestinal tract of pigs after oral ingestion of a Cyclopia genistoides (honeybush tea) extract.

Health-promoting properties such as antioxidative, anticarcinogenic, and cholesterol-lowering effects are described for mangiferin and hesperidin, the major phenolic compounds present in Cyclopia genistoides (honeybush). However, knowledge of their metabolic fate and their absorption from the gastrointestinal tract is very limited. The aim of this study was to determine the concentrations of mangiferin, hesperidin, and their metabolites in plasma, urine, and feces samples from pigs consuming an extract of Cyclopia genistoides. Pigs were administered up to 74 mg mangiferin per kilogram of body weight and 1 mg hesperidin per kilogram of body weight per day for 11 days. Plasma samples were collected at various time points on days 9 and 11 of the study and days 1 and 2 after termination of extract administration. Urine and feces were collected in fractions for 24 hours. In the plasma samples, the aglycone of mangiferin (norathyriol) was detected. Mean plasma concentrations ranged from 7.8 to 11.8 mumol/L. Six metabolites of mangiferin and hesperidin were detected in the urine, including methyl mangiferin, norathyriol, its monoglucuronide, hesperetin, hesperetin monoglucuronide, and eriodictyol monoglucuronide. Between 26.0% and 30.8% of the administered dose of hesperidin and only between 1.4% and 1.6% of mangiferin could be detected in the urine on days 9 and 11 of the study. Approximately 8.2% of the administered dose of mangiferin was determined in the feces. The main metabolite was norathyriol. Neither hesperidin nor metabolites ascribed to hesperidin intake were detected. The results suggest that formation of norathyriol from mangiferin occurs in vivo, and specific metabolites were identified in blood and excretion products in urine and feces. This study will aid in investigating the physiological functions of the parent compounds in vivo.

Development of a sample preparation method for the analysis of oxidized flavonols in onions and leek.

A method is presented for the determination of oxidized flavonols (i.e., hydroxybenzoylbenzofuranones) in plant material. Onions and leek are investigated using a newly developed solid-phase extraction procedure for sample preparation prior to high-performance liquid chromatography (HPLC). It is shown that the addition of ascorbic acid as antioxidant is necessary to prevent the generation of the researched analytes during this procedure. Nevertheless, the plant extracts do under certain conditions catalyze the oxidation of flavonoles resulting in benzofuranones.

Characterization and determination of piperine and piperine isomers in eggs.

A new analytical method for the determination of piperine and its isomers in egg yolk and albumen is described here. All four isomers were separated by HPLC and detected using UV, DAD and electrochemical detection. The absolute detection limit (UV detection, S/ N=3) of a standard solution of piperine was 370 pg piperine. The correlation coefficients for the linear calibration graphs (concentration range: c=100 ng-10 micro g piperine isomer/mL) are generally better than 0.996. The piperine isomers were characterized and identified by spectroscopy (MS, (1)H-NMR, FT-IR). The method was successfully applied to the determination of piperine deposits in eggs (egg yolk and albumen) after feeding hens with piperine-spiked feed. The detection limit for piperine (24.8(+/-0.2) ng/g egg yolk and 37.9(+/-4.9) ng/g albumen) and the recoveries (70.3(+/-7.7)% (egg yolk) and 75.7(+/-1.9)% (albumen)) of piperine were determined.