Size-exclusion High-pressure Liquid Chromatography Research Articles

Characterization of the Molecular Weight of Hyaluronan in Eye Products Using a Novel Method of Size Exclusion High-Pressure Liquid Chromatography.

Hyaluronan (HA) exists in two forms, high molecular weight HA (HMWHA) and low molecular weight HA (LMWHA), which have distinct physiological functions. Therefore it is imperative to know the form of HA within pharmaceutical products, including eye products. This study developed an accurate, sensitive, and quantitative method to characterize the form of HA in eye products. Thereafter, the effects of the HA-containing eye products on corneal wound healing were investigated. The MW distributions and concentrations of HA in over the counter eye products were determined by size exclusion chromatography (SEC) high-pressure liquid chromatography (HPLC). The effects of the eye products containing HA on corneal wound healing were characterized both in vitro and in vivo using the scratch assay and the debridement wound model, respectively. The concentrations and MWs of HA were successfully determined within a range of 0.014 to 0.25 mg/mL using SEC HPLC. The concentrations of HA in the ophthalmic products varied from 0.14 to 4.0 mg/mL and the MWs varied from ∼100 kDa to >2500 kDa. All but one HA-containing eye product had an inhibitory effect on corneal wound healing, whereas pure HA promoted corneal wound healing. A novel SEC-HPLC method was developed for quantifying and characterizing the MW of HA in eye products. Although HA promoted corneal wound healing, HA-containing eye products inhibited corneal wound healing, likely caused by preservatives. SEC-HPLC could be implemented as a routine method for determining the form of HA in commercially available ophthalmic products.

Physicochemical and Biological Stability Assessment of SB11 (Ranibizumab Biosimilar) Under Ambient and In-Use Storage for Intravitreal Administration.

SB11 (Byooviz™) is a ranibizumab biosimilar that acts as a vascular endothelial growth factor (VEGF)-A inhibitor. Stability data for unopened SB11 vials at room temperature are limited and no data are available for SB11 withdrawn into syringes (in-use) for intravitreal administration. SB11 stability was assessed in two different settings: unopened vials stored at 30 ± 2°C/65 ± 5% relative humidity (RH) for 2months, and in-use SB11 withdrawn into syringes stored at 5 ± 3°C for 98days and then 25 ± 2°C/60 ± 5% RH for 24h. The product was stored in the absence of light, and the experimental design followed International Conference on Harmonization and European Medicines Agency requirements for stability evaluation of biological products. Analysis included visual appearance (color, clarity, and presence of visible particles), pH, protein concentration (A280) and purity (size-exclusion high-pressure liquid chromatography, capillary electrophoresis-sodium dodecyl sulfate, imaged capillary isoelectric focusing), biological activity (VEGF binding and neutralization), and safety (sub-visible particulates). Except for charge variants in unopened vials at room temperature after 1month by US standards, all results met the stability acceptance criteria (US and EU) for both unopened vials and for in-use SB11. There were no major changes in terms of physicochemical stability, biological activity and sub-visible particulates. SB11 was stable for longer periods and at higher temperatures than what is stated in the labels of the reference product (Lucentis) and SB11. The physicochemical properties, biological activity, and sub-visible particulates of SB11 in both tested settings (unopened vials at room temperature and in-use product withdrawn into syringes) were maintained under the described storage periods. This information can help to avoid unnecessary delays in patient treatment without any loss in quality and biological activity, lower the workload of health care providers and reduce costs associated with drug waste.

Validation of an analytical method for the quantification of human fibrinogen in pharmaceutical products by size-exclusion liquid chromatography (SEC-HPLC).

Fibrinogen plays a vital role in normal homeostasis by promoting platelet aggregation, clot formation and fibrinolysis. It is quantified in finished pharmaceutical products using different methods described in pharmacopoeia, but these are inaccurate, difficult to validate and do not allow for identification of aggregates or protein products of the same formulation. The aim of this study was to develop and validate a method for quantification of the content of fibrinogen and other proteins present in pharmaceutical formulations by comparing it with current pharmacopeial methods. Fibrinogen was quantified in two commercial products and compared to a pharmacopeial method using a validated method for size-exclusion high-pressure liquid chromatography (SEC-HPLC). The fibrinogen level was in accordance with both products' specifications. The SEC-HPLC method showed that the percentage of fibrinogen was 94.88 for one product and 50.68 for the other, and detected high molecular weight aggregates in the second product. The SEC-HPLC method that we developed is an improvement to the current pharmacopeial method, because it allows for quantification of fibrinogen and determination of product purity. This is important because greater purity can reduce potential adverse effects of pharmaceutical products in patients.

Roadmap for the Development and Clinical Translation of Optical Tracers Cetuximab-800CW and Trastuzumab-800CW.

Optical molecular imaging using fluorescently labeled monoclonal antibodies is of significant added value in guiding surgical or endoscopic procedures. However, development of tracers for clinical trials is complex, and implementation in the clinic is therefore slow. We present a roadmap for development and translation of monoclonal antibody tracers into a drug product compliant with current good manufacturing processes (cGMPs). Methods: The production process for cetuximab-800CW and trastuzumab-800CW was optimized with regard to dye-to-protein ratio and formulation buffer. Promising formulations were produced under cGMP conditions and advanced to a full-scale stability study. Tracers were analyzed for stability by size-exclusion high-pressure liquid chromatography, pH measurement, osmolality, visual inspection, and sterility, as required by the European Pharmacopeia and cGMP guidelines. Results: Seven formulations were investigated for cetuximab-800CW and 10 for trastuzumab-800CW. On the basis of the formulation study results, we chose 2 formulations per antibody for investigation during the full-scale stability study. These formulations all performed well, showing good compliance with the acceptance criteria set for each product. Conclusion: We designed a roadmap to standardize the development, formulation, and cGMP translation of molecular fluorescent tracers. Using our standardized approach, we developed 2 stable antibody-based tracers for clinical use. The proposed roadmap can be used to efficiently develop a cGMP-compliant formulation and improve the translation of newly developed optical tracers to first-in-human use.

Organic source of vanadium. preparation and physicalchemical characteristic

The complex was obtained at room temperature: 10% water solution of SPI was mixed with 25% solution of vanadium salt (VOSO4×хH2O) in ratio 10:1 (in dry matter). The reaction was kept during 1 h at constant mixing with pH kept at 7.0- 7.1 with 1.0 M NaOH. The concentration of vanadium was determined in dry product by means of inductively coupled plasma mass-spectrometry. The chromatograms of SPI and V-SPI were obtained by means of size-exclusion high-pressure liquid chromatography, and then were integrated by weight method in the range of free till full column volume. The obtained complex of vanadium with SPI enzymatic hydrolysate (V-SPI) was water-soluble and contained 15.8 mg of vanadium per gram of product dry weight. Analysis of the molecular weight distribution of the peptide fractions of the original SPI enzymatic hydrolysate and the V-SPI complex showed that more than 87% of the vanadium complex was in peptide fractions with molecular weights more than 4.1 kD, including more than 75% of vanadium contained in fractions with molecular weights from 14.6 to 4.1 kD. The experimental evaluation in vivo will be the next stage of this research. The complex bioavailability and its effects on carbohydrate and lipid metabolism of Wistar rats with obesity will be evaluated.

Oxidation-Induced Destabilization of Model Antibody-Drug Conjugates

Oxidation of biopharmaceutics represents a major degradation pathway, which may impact bioactivity, serum half-life, and colloidal stability. This study focused on the quantification of oxidation and its effects on structure and colloidal stability for a model antibody and its lysine (ADC-L) and cysteine (ADC-C) conjugates. The effects of oxidation were evaluated by a forced degradation study using H2O2 and a shelf-life simulation, which used degrading polysorbate 80 as source for reactive oxygen species. Differential scanning fluorimetry revealed decreasing transition temperatures of the CH2 domain with rising oxidation, resulting in a loss of colloidal stability as assessed by size-exclusion high pressure liquid chromatography. The conjugation technique influences structural changes of the monoclonal antibody (mAb) and subsequently alters the impact of oxidation. ADC-C was most effected by oxidation as the CH2 domain showed the biggest destabilization on conjugation compared to the mAb and ADC-L. Quantification of Fc methionine oxidation by analytical protein A chromatography revealed 4-fold higher oxidation after 8 weeks for the ADC-C compared to the mAb. Payload degradation was observed independently of the conjugation technique used or if free in solution by ultraviolet-visible. In addition, adding antioxidants can be a suitable approach to prevent oxidation and achieve baseline stabilization of the proteins.

Composition and protein profile analysis of rice protein ingredients

The objective of this study was to investigate the nutrient composition and protein profile of a range of rice protein ingredients containing 32–78% total protein. Rice protein ingredients had significantly (P<0.05) lower levels of calcium and total essential amino acids compared to selected dairy protein ingredients, i.e., skim milk powder, whey protein isolate and whey protein hydrolysate. Protein profiles of the ingredients were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion-high pressure liquid chromatography (SE-HPLC). Since the dominant rice protein fraction (i.e., glutelin) is extensively aggregated and crosslinked through disulfide bonds, a strong reducing buffer was developed in order to solubilise the rice protein ingredients prior to analysis by SDS-PAGE. Intact rice protein ingredients (n=3) contained proteins with molecular weight (MW) ranging from ∼11 to >250kDa, while rice protein hydrolysates (n=4) were composed mainly of low MW peptides. In parallel, enriched protein fractions were extracted from defatted rice flour based on their solubility and characterised by SDS-PAGE to facilitate the identification of protein bands in the rice protein ingredients. The results of this study underpin the understanding, prediction and control of physicochemical functionality of rice protein ingredients.

Formation of protein sub-visible particles during vacuum degassing of etanercept solutions

The main purpose of this manuscript is to describe a phenomenon in which vacuum degassing a reconstituted freeze-dried fusion protein etanercept formulation caused a significant amount of protein sub-visible particles (SbVP). Physical stability of etanercept was monitored by micro-flow imaging (MFI), dynamic light scattering (DLS), size-exclusion high pressure liquid chromatography (SE-HPLC) and far- and near-ultraviolet circular dichroism (far- and near-UV CD). One potential explanation of this phenomenon is that bubble collapses when the vacuum is applied, leads to substantial heat formation, and ultimately free radical formation. Subsequently, the effect of a free-radical scavenger (ascorbic acid, AA) on SbVP formation was also evaluated. Degassing of etanercept solution by applying vacuum caused substantial increase of SbVP, as detected by MFI and DLS. However, traditional techniques such as SE-HPLC could not detect any change. The addition of free-radical scavenger had minimal effect on SbVP formation, therefore the formation of free radicals was probably not the main cause for this effect.

Early verification of myocardial ischemia with a novel biomarker of acute tissue damage: C-reactive protein fractional forms

BackgroundWe evaluated the utility of an independent biomarker of early ischemic cellular damage—circulating fractional forms of C-reactive protein (fracCRP), to verify the diagnostic relevance of low Troponin I (TnI) values within the context of a workup for Acute Coronary Syndrome (ACS). MethodsOn a semi-preparative scale, the molecular characteristics of fracCRP were established by electron microscopy and Western Blot, using isolates captured from patient serum on phosphorylcholine beads and purified by size exclusion high-pressure liquid chromatography (SE-HPLC). Captured on an analytical scale, the diagnostic utility of fracCRP was evaluated in first-draw plasma specimens (total CRP not exceeding 6mg/l) recovered from 300 cardiac emergency patients with final discharge diagnoses of ACS ruled out (N=132) or ruled in (N=168). ResultsAt a cutoff value chosen for 97.7% test specificity, the test metric (fracCRP×TnI) identified in the first blood draw 39.9% of all emergency patients ultimately diagnosed with ACS, and 17.9% of ultimately diagnosed patients who arrived with TnI values within the normal reference range (0.01–0.04ng/ml). ConclusionsThese findings suggest that the fracCRP test metric could serve as a rule-in test for ACS in a significant proportion of low to moderate risk emergency patients.

Kit for preparation of multimeric receptor-specific 99mTc-radiopharmaceuticals based on gold nanoparticles

Multivalency is a design principle by which organized arrays amplify the strength of a binding process, such as the binding of multimeric peptides to specific receptors located on cell surfaces. The conjugation of peptides to gold nanoparticles (AuNPs) produces biocompatible and stable multimeric systems with target-specific molecular recognition. The aim of this research was to develop a kit for technetium-99m (⁹⁹mTc) labelling of AuNPs that are conjugated to Lys³-bombesin, cyclo[Arg-Gly-Asp-D-Phe-Lys-(Cys)] or thiol-mannose to produce receptor-specific multimeric systems. A freeze-dried kit formulation for the instant preparation of ⁹⁹mTc-ethylenediamine-N,N'-diacetic acid (EDDA)/hydrazinonicotinyl (HYNIC)-Tyr³-octreotide (⁹⁹mTc-EDDA/HYNIC-TOC) (vial 1) and a second vial containing 1.5 ml of AuNP solution (1 nM, 20 nm diameter, surface area=1260 nm², 37,000 surface Au atoms, 1.05 × 10 particles) plus 10 µl of Lys³-bombesin, cyclo[Arg-Gly-Asp-D-Phe-Lys-(Cys)] or mannose (50 µM, approximately 285 molecules per AuNP) (vial 2) were prepared. Multimeric radiopharmaceuticals were prepared by adding 1 ml of 0.2 mol/l phosphate buffer, pH 7.0, and 1 ml of ⁹⁹mTcO4⁻ (4 GBq) to vial 1, and the mixture was incubated at 92°C for 20 min in a dry block heater. A total of 100 µl (200 MBq) of ⁹⁹mTc-EDDA/HYNIC-TOC solution (122 HYNIC-TOC molecules per AuNP) was added to vial 2. No further purification was carried out. Radiochemical purity was determined by instant thin-layer chromatography-silica gel/2-butanone (Rf values for the radiolabelled AuNP and ⁹⁹mTcO4⁻ were 0.0 and 1.0, respectively), ultrafiltration, size-exclusion high-pressure liquid chromatography and a PD-10 column. The conjugates were characterized by ultraviolet-visible, far-infrared and X-ray photoelectron spectroscopy. In-vitro binding studies were carried out in ανβ3 receptor-positive C6 glioma cancer cells, gastrin-releasing peptide receptor-positive PC3 cancer cells or mannose receptor-positive rat liver cells. Biodistribution studies were carried out in athymic mice with induced tumours (PC-3 or C6 cancer cells) or in Wistar rats (⁹⁹mTc-AuNP-mannose for sentinel lymph node detection). Images were obtained using a micro-single-photon emission computed tomography/computed tomography system. Radiochemical purity was 96 ± 2% for all of the multimeric radiopharmaceuticals. Far-infrared showed a characteristic band at 279 ± 1 cm⁻¹, which was assigned to the Au-S bond. ultraviolet-visible and X-ray photoelectron spectroscopy also indicated that the AuNPs were functionalized with peptides or mannose. Radiopharmaceuticals showed specific recognition for receptors expressed in cancer cells or rat liver cells. Micro-single-photon emission computed tomography/computed tomography images showed clear tumour uptake and lymph node accumulation. The kit (i.e. vial 1 and vial 2) demonstrated excellent stability during storage at 4°C for 6 months. Multimeric systems of ⁹⁹mTc-AuNP-peptide/mannose prepared from kits exhibited properties suitable for use as target-specific agents for molecular imaging of tumours and sentinel lymph node detection.

Abstracts of Nippon Dojo-Hiryogaku Zasshi

Abstracts of Nippon Dojo-Hiryogaku Zasshi

Bioavailability and relative tissue distribution of [125I]-recombinant human thrombin following intravenous or subcutaneous administration to non-human primates.

Recombinant human thrombin (rhThrombin) is being developed as a general adjunct to hemostasis. Endogenous thrombin is rapidly inactivated by complex formation with antithrombin III and other inhibitors. It follows that these inhibitors will also inactivate any rhThrombin that reaches the systemic circulation. Study goals were to determine the pharmacokinetic characteristics of [(125)I]-rhThrombin and [(125)I]-rhThrombin complexed to endogenous inhibitors, and the tissue distribution of rhThrombin-associated radioactivity in non-human primates. Hematology, serum chemistry and coagulation status were also monitored. [(125)I]-rhThrombin was administered intravenously (i.v.; 3.5 U kg(-1)) or subcutaneously (s.c.; 350 U kg(-1)) to male cynomolgus monkeys. Plasma was analyzed for rhThrombin-associated radioactivity and non-compartmental analysis was used to determine the corresponding pharmacokinetic parameters. A size exclusion-high pressure liquid chromatography (SE-HPLC) method was used to quantitate rhThrombin complexes, non-complexed rhThrombin, and free [(125)I]. Whole-body gamma scintigraphy was used to follow radioactivity localization up to 72 h postdose. No adverse events were observed following [(125)I]-rhThrombin administration. The pharmacokinetic profile of rhThrombin-associated radioactivity following i.v. injection was multi-exponential with an initial half-life of approximately 10 min. Following both i.v. and s.c. dosing, the terminal half-life was approximately 15 h. SE-HPLC analysis revealed that rhThrombin was rapidly complexed to antithrombin III and other inhibitors in the systemic circulation following i.v. administration. Thus, rhThrombin-associated radioactivity in the blood was complexed and presumed inactive. [(125)I]-rhThrombin inhibitor complexes accumulated and were eliminated in the liver following both routes of administration. These data suggest that rhThrombin rapidly binds to endogenous inhibitors following either i.v. or s.c. administration.

Closomers of High Boron Content: Synthesis, Characterization, and Potential Application as Unimolecular Nanoparticle Delivery Vehicles for Boron Neutron Capture Therapy

Unique nanosized closomers of high boron content that may exhibit potential as boron neutron capture therapy target species have been synthesized. The design of these boron-rich nanospheres is based in part on previous work involving dodeca(carboranyl)-substituted closomers [Thomas, J.; Hawthorne, M. F. Chem. Commun. 2001, 1884-1885]. Coupling of ortho-carborane moieties through ester and ether linkages to the rigid [closo-B(12)(OH)(12)](2-) scaffold resulted in the development of a 12(12)-closomer-ester derivative, dodeca[6-(1,2-dicarba-closo-dodecaboran-1-yl)hexanoate]-closo-dodecaborate (2-), 6, and 12(12)-closomer-ether derivatives, dodeca[6-(2-methy1-1,2-dicarba-nido-dodecaboran-1-yl)hexyl]-closo-dodecaborane (14-), 14, and dodeca[6-(7,8-dicarba-nido-dodecaboran-7-yl)hexyl]-closo-dodecaborane (14-), 15. These closomers were investigated by UV-visible spectroscopy and cyclic voltammetry. Additionally, a deboronation method employing NaCN as the nucleophilic reagent was utilized to obtain sodium salts of the ether-linked nido-closomer polyanions, which were purified using a newly developed size-exclusion high pressure liquid chromatography method.

Detection of secretory IgM in tears during rhino-conjunctivitis

With various methods, secretory immunoglobulin M (sIgM) was assessed in tears of patients with rhino-conjunctivitis. Tears were analyzed by microimmunoelectrophoresis (MIE), size-exclusion high-pressure liquid chromatography (SE-HPLC), sodium dodecyl sulphate (SDS) electrophoresis and isoelectric focusing. Only very small traces of serum IgM could be found in tears of healthy volunteers. MIE showed that tear sIgM is different from serum IgM. The former migrates in the direction of the anode, while serum IgM migrates to the cathode. The SDS electropherogram of a number of patients showed an additional strong band of the sIgM mu-chain of approximately 68 kDa, after rhino-conjunctivitis. SE-HPLC showed two additional peaks for these patients, at 10.97 min and at 12.94 min, which were attributed to tear sIgM. The former peak consists of a complex of four sIgM molecules. One year later the chromatograms of the former rhino-conjunctivitis patients did not show any peak of sIgM in SE-HPLC. Serum IgM of a molecular weight (MW) 970 kDa appears only in normal tears at very low concentrations, as a result of transudation from the serum. In contrast, sIgM is synthesized during rhino-conjunctivitis in high concentrations in the human lacrimal glands and the conjunctiva. These increased levels of tear sIgM are due to stimuli for specific protein synthesis.

Asymmetrical flow field-flow fractionation and multiangle light scattering for analysis of gelatin nanoparticle drug carrier systems.

The physicochemical properties of nanosized colloidal drug carrier systems are of great influence on drug efficacy. Consequently, a broad spectrum of analytical techniques is applied for comprehensive drug carrier characterization. It is the primary objective of this paper to present asymmetrical flow field-flow fractionation (AF4), coupled online with multiangle light scattering detection, for the characterization of gelatin nanoparticles. Size and size distribution of drug-loaded and unloaded nanoparticles were determined, and data were correlated with results of state-of-the-art methods, such as scanning electron microscopy and photon correlation spectroscopy. Moreover, the AF4 fractionation of gelatin nanoparticulate carriers from a protein model drug is demonstrated for the first time, proposing a feasible way to assess the amount of loaded drug in situ without sample preparation. This hypothesis was set into practice by monitoring the drug loading of nanoparticles with oligonucleotide payloads. In this realm, various fractions of gelatin bulk material were analyzed via AF4 and size-exclusion high-pressure liquid chromatography. Mass distributions and high-molecular-weight fraction ratios of the gelatin samples varied, depending on the separation method applied. In general, the AF4 method demonstrated the ability to comprehensively characterize polymeric gelatin bulk material as well as drug-loaded and unloaded nanoparticles in terms of size, size distribution, molecular weight, and loading efficiency.

Lysozyme: a mediator of myocardial depression and adrenergic dysfunction in septic shock in dogs

The objective of the present study was to identify the nature of a filterable cardiodepressant substance (FCS) that contributes to myocardial dysfunction in a canine model of Escherichia coli septic shock. In a previous study, it was found that FCS increased in plasma after 4 h of bacteremia (Am J Physiol 1993;264:H1402) in which FCS was identified by a bioassay that included a right ventricular trabecular (RVT) preparation. In that study, FCS was only partially identified by pore filtration techniques and was found to be a protein of molecular weight between 10 and 30 K. In the present study, FCS was further purified by size exclusion high-pressure liquid chromatography, until a single band was identified on one-dimensional gel electrophoresis. This band was then subjected to tandem mass spectrometry and protein-sequencing techniques and both techniques identified FCS as lysozyme c (Lzm-S), consistent with that originating from the canine spleen. Confirmatory tests showed that purified Lzm-S produced myocardial depression in the RVT preparation at concentrations achieved during sepsis in the in vivo preparation. In addition, Lzm-S inhibited the adrenergic response induced by field stimulation and the β - agonist isoproterenol in in vitro preparations, these results suggesting that Lzm-S may inhibit the sympathetic response in sepsis. The present findings indicate that Lzm-S originating from disintegrating leukocytes from organs such as the spleen contributes to myocardial dysfunction in this model. The mechanism may relate to its binding or hydrolysis of a cardiac membrane glycoprotein thereby interfering with myocardial excitation–contraction coupling in sepsis.

Equilibrium denaturation studies of the Escherichia coli factor for inversion stimulation: implications for in vivo function.

The Factor for Inversion Stimulation (FIS) is a dimeric DNA binding protein found in enteric bacteria that is involved in various cellular processes, including stimulation of certain specialized DNA recombination events and transcription regulation of a large number of genes. The intracellular FIS concentration, when cells are grown in rich media, varies dramatically during the early logarithmic growth phase. Its broad range of concentrations could potentially affect the nature of its quaternary structure, which in turn, could affect its ability to function in vivo. Thus, we examined the stability of FIS homodimers under a wide range of concentrations relevant to in vivo expression levels. Its urea-induced equilibrium denaturation was monitored by far- and near-UV circular dichroism (CD), tyrosine fluorescence, and tyrosine fluorescence anisotropy. The denaturation transitions obtained were concentration-dependent and showed similar midpoints (C(m)) and m values, suggesting a two-state denaturation process involving the native dimer and unfolded monomers (N(2) <--> 2U). The DeltaG(H(2)O) for the unfolding of FIS determined from global and individual curve fitting was 14.2 kcal/mole. At concentrations <9 microM, the FIS dimer began to dissociate, as noted by the change in CD signal and size-exclusion high-pressure liquid chromatography retention times and peak width. The estimated dimer dissociation constant based on the CD and size-exclusion chromatography data is in the micromolar range, resulting in a DeltaG(H(2)O) of at least 5 kcal/mole less than that calculated from the urea denaturation data. This discrepancy suggests a deviation from a two-state denaturation model, perhaps due to a marginally stable monomeric intermediate. These observations have implications for the stability and function of FIS in vivo.

Effect of Flour Water-Extractable Pentosans on Molecular Associations in Gluten During Mixing

Wheat gluten and mixtures of gluten and water-extractable pentosans (WEP) were mixed in a counter-rotating batch mixer at constant hydration level, in order to study the effects of feruloylated arabinoxylans (AX) on gluten mixing properties. The presence of WEP delayed the development time of gluten, suggesting a competition for water during the first stage of dough formation. Changes in macromolecular associations were followed using size-exclusion high-pressure liquid chromatography (SE-HPLC), SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and solubility measurements. WEP did not affect the breakdown rate of proteins during mixing, when compared on specific energy mechanical basis. Mixing did not affect AX solubility and viscosity. Nevertheless, ferulic acid (FA) content decreased during mixing. This decrease involved mostly FA esters of compounds soluble in acetone/methanol/water (7/7/6, v/v/v) which are present in gluten. These soluble FA esters are likely to react with proteins during mixing. They would be involved, rather than feruloylated AX, in gluten breakdown during overmixing, since no covalent complex between AX and protein was detected in overmixed doughs.

Preservation of protein in marine systems: Hydrophobic and other noncovalent associations as major stabilizing forces

—The fate of proteins during early diagenesis was investigated in environments with low mineral content to assess preservation mechanisms other than mineral sorption. Preservation was examined in anoxic, organic-rich sediments of Mangrove Lake, a marine environment located in Bermuda, and for particulate material generated during oxic decay of diatoms. N-phenacylthiazolium bromide (PTB) treatment tested the hypothesis that proteins may undergo modification reactions with glucose to form advanced-glycation end products (AGEs). A small but significant release (additional 14%) of proteins was observed after PTB treatment in surficial sediments, indicating that some aggregations can proceed through an α-dicarbonyl intermediate of the AGE pathway. Size-exclusion high-pressure liquid chromatography with protein fluorescence, absorbance, and evaporative light-scattering detector measurements under native (phosphate or bicarbonate buffers) and denaturing (guanidine · HCl, urea, or acetonitrile) conditions point to the importance of hydrophobic and other noncovalent interactions in the stabilization of proteinaceous material in the environment. Soluble aggregates of substantial, relative molecular mass ( M r ≳ 10 6) appear to be formed early in the diagenetic sequence. The preferential preservation of very high M r, multisubunit phytoplankton proteins in sediments suggests that such aggregations confer resistance to degradation. Alternatively, some of the proteinaceous material may represent that fraction of organic matter that is highly prone to aggregations. Extended incubations (18 h; 37°C) with trypsin and proteinase-K showed that much of the aggregates that could be extracted are receptive to proteolytic cleavage. Buffer-, surfactant-, and NaOH-extractable aggregates comprised most of the acid-hydrolyzable proteinaceous material in detritus and surficial sediments but <35% in 9.7-m-deep sediments, suggesting additional mechanisms for preservation might be in operation. The results are direct evidence for the preservation of peptide linkages in sediments as old as 4000 yr and for noncovalent associations (hydrophobic interaction, hydrogen bonding) of protein as important mechanisms in long-term preservation.

Testing and comparing oxidative stability of vegetable oils and fats at frying temperature

A new method has been developed to estimate the stabilising activity of synthetic and natural food additives at frying. Non-refined and refined vegetable fats and oils were heated at a temperature of 170°C after adding water-conditioned silica gel for two hours. The degraded products were measured to assess the oil stability at frying temperature. The determination of polymeric triglycerides by size exclusion high-pressure liquid chromatography (HPLC) was carried out for the estimation of the oxidative heat stability of vegetable fats and oils. Tocopherols, various tocopherol esters and phytosterol fractions, phenolic compounds, like quercetin, oryzanol, ferulic acid, squalene, butyl hydroxytoluol (BHT), butyl hydroxyanisol (BHA), and other compounds, like ascorbic acid 6-palmitate and gallates, are added to refined sunflower and rapeseed oil and their efficacy determined. Both linoleic and oleic rich oils gave comparable results for the activity of the various compounds. α-tocopherol, tocopherol esters, and BHA have low effects at frying temperature. Ascorbic acid 6-palmitate and some phytosterol fractions were found to have the greatest antioxidant activity. Corn oil was more stable than soybean oil and rapeseed oil better than olive oil. It was also observed that non-refined oils proved to have a better stability at elevated temperature than refined oils. The results show that the stability of the vegetable oils at frying temperature is a function of more than just the fatty acid composition. There is evidence which supports a co-relationship between the unsaponifiable matter content and oxidative stability. It is believed that a radical peroxidation mechanism predominates at lower temperatures. When a large volume of oil is heated in a fryer and the oxygen supply is poor, non-radical reactions such as elimination (acid catalysed dehydration) or nucleophilic substitution take place.