A colorimetric method for determination of γ-glutamyl-S-ethenyl-cysteine in narbon vetch (Vicia narbonensis L.) seeds.

A new colorimetric method based on the bleaching of the iodoplatinate ion has been developed for fast and easy determination of γ-glutamyl-S-ethenyl-cysteine (GEC) in narbon vetch (Vicia narbonensis L.) seeds. The calibration curve showed a good correlation (r(2)=0.9959) between absorbance and GEC amounts from 5.5 to 33 µg (10-59.78 µmol/L). The limits of detection and quantification were 1.16 and 3.55 µmol/L, respectively, and no significant interferences from other sulfur-containing compounds were observed. The method showed excellent repeatability (relative standard deviation [RSD]=0.28%), reproducibility (RSD=4.4%), and accuracy (94%). Determination of GEC in 20 narbon vetch accessions yielded values that were in agreement with those reported previously using capillary electrophoresis and high-performance liquid chromatography methods. The method could be especially valuable for determination of GEC during the process of production of new low-GEC narbon vetch varieties.

An interlaboratory study as useful tool for proficiency testing of chemical oxygen demand measurements using solid substrates and liquid samples with high suspended solid content.

In 2008, the first Proficiency Testing Scheme of Chemical Oxygen Demand (1(st)COD-PT(ADG)) was conducted to assess the results obtained for different research groups whose field work is mainly anaerobic digestion. This study was performed using four samples, two solid samples as raw materials and two solid samples to prepare high concentration suspended solid solutions. Invitations were sent to a large number of laboratories, mainly to anaerobic digestion research groups. Finally, thirty labs from sixteen countries agreed to participate, but for different reasons four participants could not send any data. In total, twenty-six results were reported to the COD-PT coordinator. This study showed the importance of continuous participation in proficiency testing (PT) schemes in order to compare the results obtained. Taking into account the lack of a general standard method and high quality certified reference materials (CRMs), the traceability of COD determination is not currently easy to check. In addition, the spread of participants' results obtained was high and pointed to the advisability of using consensus values due to their unreliability. Therefore, the theoretical oxygen demand (ThOD) values were considered as assigned values for all the samples analysed. On the other hand, in this PT the established standard deviation (ESD) has been determined by the Horwitz modified function. Participants of this 1(st)COD-PT(ADG) were asked to give a short report on the analytical method used. Although all the participants used potassium dichromate as their oxidant reagent, their experimental procedures were very different. With the purpose of comparing the results obtained, the different experimental conditions used were classified into five methods, corresponding to two main categories, open and closed reflux. The performance of laboratories was expressed by the z-score, whose value is considered satisfactory when z-score

Assessment of a modified and optimised method for determining chemical oxygen demand of solid substrates and solutions with high suspended solid content.

A modified approach to determine the chemical oxygen demand (COD) of solid substrates based on the DIN 38414-S9 standard method is proposed. The adapted procedure is assessed and compared with standard methods widely used for water and wastewater such as the American Public Health Association-American Water Works Association-Water Pollution Control Federation (APHA-AWWA-WPCF) standard methods 5220 B-open reflux (SM-OR) and 5220 D-closed reflux colorimetric (SM-CR). Solutions with high suspended concentration of solids, as well as digestates from an anaerobic reactor, were used during the comparative test. For solid substrates, the COD recovery was about 100% when the proposed method was used. For solutions with solid content higher than 20 g TS L(-1), the recovery was only completed when the proposed method was used, showing that the methods traditionally employed are not very appropriate for samples with the described characteristics. For instance, percentages of COD recovery in the ranges of 77.3-87.1% and 89.4-94.1% were achieved when the SM-OR and SM-CR methods were used, respectively.

Amino acids composition of Teucrium nutlet proteins and their systematic significance.

BACKGROUND AND AIMS: Plant species are considered as a good source of dietary proteins, although the nutritional quality of proteins depends on their amino acid composition. In this work the protein content and amino acid composition of nutlets of 21 Teucrium taxa (Lamiaceae) from Spain were analysed and their nutritional quality was compared with the minimum values established by the Food and Agriculture Organization of the United Nations (FAO). In addition, the amino acid composition was evaluated as a chemical character to clarify the taxonomic complexity in this genus. METHODS: Amino acid content of nutlets was determined after derivatization with diethyl ethoxymethylenemalonate by high-performance liquid chromatography. Previously, nutlets samples were hydrolysed and incubated in an oven at 110 degrees C for 24 h. KEY RESULTS: The protein content was variable, ranging from 6.4 % in T. dunense to 43.8 % in T. algarbiense. According to the FAO values all taxa contain satisfactory amounts of leucine, threonine and valine and are deficient in lysine. The similarity analysis of Teucrium taxa using amino acid composition data did not clearly reflect the infrageneric classification of this genus. CONCLUSIONS: Annual species, such as T. spinosum, T. aristatum and T. resupinatum showed a better balanced amino acid composition. The dendrogram partly matched with the karyological complexity of Teucrium. No correlation between amino acid composition and habitat has been observed, showing that Teucrium nutlet amino acid composition may not be strongly influenced by the environment.

Influence of cultivar and fruit ripening on olive (Olea europaea) fruit protein content, composition, and antioxidant activity.

Proteins of olive fruit mesocarp are not very well-known at present. However, they have been shown to pass, at least partially, to the olive oil during its elaboration and therefore might be contributing to some of the special characteristics of this vegetable oil. In this study, protein content and composition were determined in olive fruits, cv. Arbequina and Picual, at three stages of ripening: green, spotted, and purple. Mesocarp proteins constituted 1.3-1.8% of the dry weight of the olive fruit, and cultivar and fruit ripening did not produce important changes in mesocarp protein content or composition. In addition, this composition was also similar to the amino acid composition of a 4.6-kDa polypeptide, which is the major protein component of olive oils and of oil bodies of olive fruit mesocarp, suggesting that this polypeptide is likely to be a major component of mesocarp proteins. There was, also, a relationship between the oil content of the olive fruit and the protein content determined, suggesting a stabilizing function of these proteins in the oil bodies of the olive fruit, analogously to the role suggested for oleosins. This stabilizing function does not seem to be extended to olive oils because when the polypeptides isolated were added at 20 ppm to soybean oil, the stability of the oil increased only slightly, suggesting that if these compounds play some role in the stability of the oils, this should be mostly a consequence of the possible interactions among these protein components and other olive oil antioxidant constituents.

Pyrrolization and antioxidant function of proteins following oxidative stress.

The consequences of oxidative stress on microsomal proteins were analyzed by studying their pyrrolization and the antioxidative activity of the modified proteins produced. The microsomal system consisted of freshly prepared trout muscle microsomes, which were oxidized in the presence of 5 microM Cu(2+), 1 mM Fe(3+)/5 mM ascorbate, or 1 mM Cu(2+)/10 mM H(2)O(2). Pyrroles on proteins were detected by forming Ehrlich adducts with p-(dimethylamino)benzaldehyde and by determination of epsilon-N-pyrrolylnorleucine (Pnl) by capillary electrophoresis. Their antioxidative activity was studied by testing two model pyrrolized proteins (dimeric and monomeric modified bovine serum albumin: DBSA and MBSA, respectively), which were produced in the reaction of BSA and 4,5(E)-epoxy-2(E)-heptenal. These proteins were assayed at a concentration of 10-40 microg/mL, which was selected because at this concentration both DBSA and MBSA had a concentration of Pnl similar to the Pnl concentration produced in oxidized microsomes. Both DBSA and MBSA significantly (p < 0.05) protected against lipid peroxidation, assessed by the formation of thiobarbituric acid reactive substances (TBARS), and protein damage, evaluated by amino acid analysis, for the three systems assayed, and this protection was always higher than that exhibited by BSA, which was used as control. The order of effectiveness was DBSA > MBSA > BSA and was parallel to the Pnl content in the assayed proteins. These results suggest that antioxidative activity of BSA may also be related to its ability to react with lipid oxidation products and to produce modified BSA with antioxidative activity. This mechanism may also be contributing to the antioxidative activity exhibited by many proteins.

Determination of peptides and proteins in fats and oils.

A method for the determination of proteins in fats and oils is described. Proteins were sequentially precipitated with acetone and hydrolyzed, and the produced amino acids were fractionated and quantificated. This analysis protocol afforded a method of high sensitivity and specificity which was fully evaluated and validated. The data obtained showed good accuracy and linearity with excellent reproducibility and recovery. When the method was applied to 40 olive oils, all of them contained proteins in the range 10-50 microg/100 g of oil, suggesting that proteins are nonpreviously described minor components of these oils. In addition, the proteins precipitated were almost exclusively composed by one polypeptide of apparent 4600 molecular weight, which was isolated from olive drupes and partially characterized by amino acid analysis. Similar polypeptides were also detected in other seeds, suggesting that they may constitute a new class of polypeptides in plants with oleosin-like characteristics. Furthermore, the method was also applied to different fats and oils, and all the samples analyzed contained proteins, suggesting that natural fats and oils always contain polypeptides and/or proteins as minor components. These results also suggest that some peptides are soluble in lipid matrixes, where they might be playing unknown functions. The developed procedure provides a methodology for the determination of these components.

Contribution of pyrrole formation and polymerization to the nonenzymatic browning produced by amino-carbonyl reactions.

Recent studies have hypothesized that pyrrole formation and polymerization may be contribute to the nonenzymatic browning produced in both oxidized lipid/protein reactions and the Maillard reaction. To develop a methodology that would allow investigation of the contribution of this browning mechanism, the kinetics of formation of color, fluorescence, and pyrrolization in 4, 5(E)-epoxy-2(E)-heptenal/lysine and linolenic acid/lysine model systems were studied. In both cases similar kinetics for the three measurements were observed at the two temperatures assayed (37 and 60 degrees C), and there was a high correlation among color, fluorescence, and pyrrolization measurements obtained as a function of incubation time. Because the color and fluorescence production in the 4,5(E)-epoxy-2(E)-heptenal/lysine system is a consequence of pyrrole formation and polymerization, the high correlations observed with the unsaturated fatty acid also suggest a contribution of the pyrrole formation and polymerization to the development of color and fluorescence observed in the fatty acid/lysine system. Although the contribution of other mechanisms cannot be discarded, all of these results suggest that when the pyrrole formation and polymerization mechanism contributes to the nonenzymatic browning of foods, a high correlation among color, fluorescence, and pyrrolization measurements should be expected.

Determination of epsilon-N-pyrrolylnorleucine in fresh food products.

epsilon-N-Pyrrolylnorleucine was determined in different fresh food products to study its presence as a normal component of food proteins. Twenty-two different products were screened: cod, cuttlefish, salmon, sardine, trout, beef, chicken, pork, broad bean, broccoli, chickpea, garlic, green pea, lentil, mushroom, soybean, spinach, sunflower, almond, hazelnut, peanut, and walnut. Foods were homogenized, their proteins were precipitated with trichloroacetic acid and hydrolyzed with 2 N NaOH for 20 h, and the epsilon-N-pyrrolylnorleucine content was determined by capillary electrophoresis. The epsilon-N-pyrrolylnorleucine, which was identified by HPLC/MS in sardine muscle hydrolysate, ranged in the 22 foods analyzed from 0.24 to 6.36 micromol/g. This concentration was correlated with the protein content of the food (r = 0.687, p = 0.00041). In addition, the epsilon-N-pyrrolylnorleucine/lysine ratio was found to be a function of the lipid, iron, and protein contents of the food (r = 0.881, p < 0.0001) and was directly correlated with lipid and iron contents and inversely correlated with the protein content. These results are in agreement with the oxidative stress origin proposed for epsilon-N-pyrrolylnorleucine and suggest that the epsilon-N-pyrrolylnorleucine/lysine ratio is a characteristic of each food. In addition, epsilon-N-pyrrolylnorleucine seemed to be a normal component of many fresh food products, in which it may be acting as a natural antioxidant.

Effect of pH and temperature on comparative nonenzymatic browning of proteins produced by oxidized lipids and carbohydrates.

Bovine serum albumin (BSA) was incubated for 24 h in the presence of 10 mM ribose (RI), methyl linoleate hydroperoxides, or the secondary products of methyl linoleate oxidation (SP), at five temperatures (25, 37, 50, 80, and 120 degrees C) and different pHs (4, 7, and 10), to study the influence of these variables in the browning, fluorescence, amino acid losses, and pyrrolization of the modified proteins. All treated proteins exhibited similar colors and fluorescence spectra, and the spectra of their Ehrlich adducts were also analogous. However, at 25-50 degrees C the proteins treated with oxidized lipids exhibited higher color changes, amino acid losses, and pyrrolization than the BSA treated with RI, and these effects were much higher in proteins treated with RI at 80-120 degrees C. The effect of pH was similar in proteins treated with RI or SP. These results suggested a similarity for browned proteins obtained from both carbohydrates and oxidized lipids. In addition, both reactions seem to be complementary, because melanoidin formation derived from oxidized lipids can be produced under conditions different from those carbohydrate/protein reactions.

Effect of pH and temperature on comparative antioxidant activity of nonenzymatically browned proteins produced by reaction with oxidized lipids and carbohydrates.

The antioxidative activity of nonenzymatically browned bovine serum albumin (BSA) produced by reaction with ribose (RI), hydroperoxides of methyl linoleate oxidation (HP), and secondary products of methyl linoleate oxidation (SP), at different pHs (4, 7, and 10) and temperatures (25, 37, 50, 80, and 120 degrees C), was studied to compare the antioxidative effects of carbohydrate- and oxidized lipids-modified proteins. The modified proteins (RIBSA, HPBSA, and SPBSA) were tested for antioxidative activity (at 100 ppm) in soybean oil using the thiobarbituric acid-reactive substances (TBARS) assay. All of them decreased significantly (p < 0.05) the TBARS formation in the oil and exhibited different effectiveness as a function of the temperature and the pH of the medium. In addition, there was a good correlation between the antioxidative activity of the protein and the amino acid losses produced during the nonenzymatic browning. These results are in agreement with an analogous and complimentary contribution of both Maillard and oxidized lipid/protein reactions to the antioxidative activity produced in foods during processing and storage.

Modification of histidine residues by 4,5-epoxy-2-alkenals.

The reactions of 4,5(E)-epoxy-2(E)-heptenal with 4-methylimidazole and N(alpha)-acetyl-L-histidine methyl ester were studied to characterize the adducts produced in the modification of histidine residues by epoxyalkenals and to develop a methodology for the determination of these adducts in protein hydrolysates. The reaction products, which were isolated and characterized, resulted in the Michael adducts produced in the addition of one of the imidazolic nitrogens to the carbon-carbon double bond of the epoxyalkenal. Only some of the theoretical isomers were produced. Thus, in the reaction with 4-methylimidazole, the main product was 4, 5-epoxy-3-(4-methylimidazol-1-yl)heptanol (88%), although the formation of 4,5-epoxy-3-(5-methylimidazol-1-yl)heptanol (12%) was also observed. On the other hand, the reaction with N(alpha)-acetyl-L-histidine methyl ester produced exclusively N(alpha)-acetyl-1-[1'-(1' ',2' '-epoxybutyl)-3'-hydroxypropyl]-L-histidine methyl ester. This last compound was used to develop a procedure for the determination of 4, 5(E)-epoxy-2(E)-heptenal-histidine adducts in protein hydrolysates. When this procedure was applied to the analysis of bovine serum albumin treated with 0.01-10 mM 4,5(E)-epoxy-2(E)-heptenal, the formation of the adduct was observed and its concentration increased with the concentration of the aldehyde and the incubation time, and was parallel to the histidine losses observed in the protein after acid hydrolysis as well as to the formation of protein carbonyls. In addition, the number of histidine residues lost in the protein was very similar to the number of adduct residues produced, suggesting that the addition reaction is the major mechanism for histidine losses suffered by proteins following their reaction with epoxyalkenals.

A spectrophotometric method for the determination of proteins damaged by oxidized lipids.

The Ehrlich reaction was optimized to determine pyrrolized proteins produced as a consequence of lipid peroxidation and oxidative stress. The procedure consisted of the treatment of the modified protein with p-(dimethylamino)benzaldehyde at a controlled acidity and temperature, and the determination of adducts produced against the blank obtained in the absence of the reagent. The extinction coefficient of Ehrlich adducts was calculated by using epsilon-N-pyrrolylnorleucine (Pnl) as standard and was 35,000 M-1 cm-1. The response was linear and reproducible within the range 0. 16-20 microM Pnl. The assay was applied to determination of pyrrole content in bovine serum albumin, bovine alpha-globulins, bovine gamma-globulins, and mixtures of them, incubated overnight with 1 mM of 4,5(E)-epoxy-2(E)-heptenal, obtaining results similar to those from determination of Pnl by capillary electrophoresis after basic hydrolysis of the protein. The method was also applied to pyrrole determination in bovine plasma proteins either incubated with epoxyalkenals, hydroxyalkenals, lipid hydroperoxides, and secondary products of lipid peroxidation, or oxidized with Fe3+/ascorbate. All these treatments produced pyrrolization of plasma proteins and all Ehrlich adducts gave very similar absorbance spectra with the exception of that produced in the treatment with hydroxyalkenals. The above results suggest that protein pyrrolization is a normal consequence of the lipid peroxidation process and of oxidative stress, and that Ehrlich adducts may be valid to determine this pyrrolization.

Feed-back inhibition of oxidative stress by oxidized lipid/amino acid reaction products.

Three oxidized lipid/amino acid reaction products (OLAARPs): 1-methyl-4-pentyl-1,4-dihydropyridine-3,5-dicarbaldehyde, 1-(5-amino-1-carboxypentyl)pyrrole, and N-(carbobenzyloxy)-1(3)-[1-(formylmethyl)hexyl]-l-histidine dihydrate, were prepared and tested for antioxidative activity in a microsomal system in order to investigate the effect that OLAARP formation may be playing in the oxidative stress process. The microsomal system consisted of freshly prepared trout muscle microsomes, which were oxidized in the presence of 5 microM Cu2+, 1 mM Fe3+/5 mM ascorbate, or 1 mM Cu2+/10 mM H2O2, and the compound to be tested as antioxidant added at 50 microM. At different periods of time, samples were tested for lipid peroxidation, assessed by the formation of thiobarbituric acid reactive substances (TBARS), and protein damage, which was evaluated by the formation of protein carbonyls and amino acid analysis. The three OLAARPs and butylated hydroxytoluene significantly (p < 0.05) protected against lipid peroxidation and protein damage for the three systems assayed. On the contrary, neither the amino acids used in the preparation of OLAARPs nor alpha-tocopherol, mannitol, aminoguanidine, or 4, 5-dihydroxy-1,3-benzenedisulfonic acid exhibited this constant protection. Because OLAARPs were produced at inhibitory levels during microsomal lipid peroxidation, these results suggest that OLAARP formation may be an antioxidative defense mechanism by which oxidative stress is feed-back-inhibited, delaying the damage caused by reactive oxygen species.

Changes induced in bovine serum albumin following interactions with the lipid peroxidation product E-2-octenal.

Bovine serum albumin (BSA) undergoes a number of deteriorative changes when exposed to E-2-octenal. Reaction of BSA with E-2-octenal produced fluorescent BSA with an excitation maximum at 350 nm and emission maximum at 440 nm and promoted polymerization. Amino acid analysis of the modified BSA showed that the E-2-octenal treatment leads to the selective loss of lysine residues and the formation of new amino acid derivatives. The same products were detected in acid hydrolysates of poly-L-lysine and N2-(carbobenzyloxy)-L-lysine after their reactions with E-2-octenal. The reaction of N2-(carbobenzyloxy)-L-lysine with E-2-octenal led to the production of 1-[N2-(carbobenzyloxy)-L-lysyl]-2- (1'-carboxymethyl)-4-pentylpyridinium betaine, 1-[N2-(carbobenzyloxy)-L-lysyl]-2- (3'-carboxy-2'-E-propen-1'-yl)-4- pentylpyridinium betaine and bis[1-[N2-(carbobenzyloxy)-L-lysyl]-2- (3'-carboxy-2'-propen-1',2'-diyl)- 4-pentylpyridinium betaine] (isomeric mixture). Upon acid hydrolysis, these quaternary pyridinium salts led to new amino acid derivatives, presumably 1-(L-lysyl)-2-(1'-carboxymethyl)-4-pentylpyridinium betaine, 1-(L-lysyl)-2-(3'-carboxy-2'-E-propen-1'-yl)- 4-pentylpyridinium betaine and bis[1-(L-lysyl)-2-(3'-carboxy-2'-propen-1',2'-diyl)- 4-pentylpyridinium betaine] (isomeric mixture) that were indistinguishable from those obtained from BSA, poly-L-lysine and N2-(carbobenzyloxy)-L-lysine after similar treatment. The reaction of lysine residues with E-2-octenal provides the basis for methods by which the contributions of E-2-octenal in the modifications of proteins can be determined.

Modification of delipidated apoprotein B of low density lipoprotein by lipid oxidation products in relation to macrophage scavenger receptor binding.

It is known that macrophages recognize and take up oxidized low density lipoprotein (LDL) and this macrophage recognition has been suggested to be due to modification of the lysine (Lys) residues of apoprotein B (apo B). In order to determine whether such modification is involved in recognition, delipidated apo B was modified with lipid peroxidation products and the macrophage recognition of the modified apo B was examined. When delipidated apo B was treated with linoleic acid 13-mono-hydroperoxide (LOOH) and trans-2-octenal (octenal), apo B became fluorescent and its Lys and histidine (His) residues were decreased. When delipidated apo B, partially methylated at the epsilon-amino groups of Lys residues, was treated with LOOH and octenal, fluorescence was not produced and the free Lys residues were not decreased. LOOH- and octenal-modified delipidated apo B were recognized by mouse peritoneal macrophages. The macrophage recognition of the modified apo B was prevented by maleyl bovine serum albumin, indicating that the scavenger receptor was involved in recognition. Neither methyl apo B nor methyl apo B, modified with LOOH or octenal, was recognized by macrophages. Neutralization of positively charged Lys residues of apo B by modification with LOOH and octenal may be requisite for recognition. Bovine serum albumin modified with LOOH and octenal prevented the recognition of the modified apo B, indicating that none of the intrinsic structure of apo B is required for recognition.

High-performance liquid chromatographic determination of N-epsilon-(2-propenal)lysine in biological samples after derivatization with diethylethoxymethylenemalonate.

A recently reported methodology for amino acid analysis by HPLC has been adapted for quantification of N-epsilon-(2-propenal)lysine (a modified lysine by reaction with malondialdehyde that has been found in enzymatic digests of foods and in urine) in biological samples. We describe its use for investigating the in vitro degradation of N-epsilon-(2-propenal)lysine using rat tissue homogenates. Lysine dipeptide, used as a control in the incubation mixtures, and the lysine released by the hydrolytic action of the homogenates in the in vitro incubations are quantified in the same way. The samples are subjected to a cleanup prederivatization step using PD-10 disposable columns (Pharmacia). This allows precolumn derivatization with diethylethoxymethylenemalonate (50 min, 50 degrees C) and resolution of the derivatives of the compounds of interest by reversed-phase HPLC (binary gradient, 45 min) with quantification based on the uv absorption of the derivatives at 280 nm (detection limits below 1 pmol). The entire analysis takes 110 min. This method can be of general use for the determination of N-epsilon-(2-propenal)lysine in the context of research dealing with protein deterioration by reaction with malondialdehyde in biological systems and in foods. A method for the synthesis of N-epsilon-(2-propenal)lysine, used as external standard for the HPLC analysis, is described.

Amino acid analysis by high-performance liquid chromatography after derivatization with diethyl ethoxymethylenemalonate.

Amino acids were determined by precolumn derivatization with diethyl ethoxymethylenemalonate and reversed-phase high-performance liquid chromatography (HPLC) with spectrophotometric detection at 280 nm. The reaction time was 50 min and the derivatives were stable at room temperature. Chromatographic resolution of a mixture of the derivatives of seventeen amino acids, including proline and cystine, was achieved within 35 min using a binary gradient system. The detection limit was 3 pmol. Amino acid analyses of acid hydrolysates of two proteins gave results equivalent to those obtained by conventional ion-exchange-based amino acid analysis. The simplicity of the procedure allows its use on any multi-purpose HPLC system.