Collagens made from giant salamander (Andrias davidianus) skin and their odorants.

Two collagens were made from giant salamander (Andrias davidianus) skin by using acid and pepsin extraction methods. The yields of acid-soluble and pepsin-soluble collagens were 26.9 and 58.7%, respectively. The results of spectrum, electrophoresis and amino acid analysis showed that they were type 1 collagen with two α and one ß peptides and high imino acid content. They had low solubility at a pH above 6 or salt concentration over 5%. The pepsin-soluble collagen had a better emulsion activity index. The odorants in raw skin and collagens were identified and evaluated using gas-chromatography mass-spectrometer and olfactometry methods and sensory analysis. The fishy and fatty off-odors in skin were not perceivable in the collagens. Sour, ammonia-like, and acrid off-odors were found in the collagens due to acid and enzymatic hydrolysis and protein degradation. The off-odor intensity of pepsin-soluble collagen was low. It could be considered a good and safe collagen material.

Using D- and L-Amino Acid Oxidases to Generate the Imino Acid Substrate to Measure the Activity of the Novel Rid (Enamine/Imine Deaminase) Class of Enzymes.

This chapter describes a method to assay the activity of reactive intermediate deaminases (Rid), a large family of conserved soluble enzymes, which have been proposed to prevent damages from metabolic intermediates such as the highly reactive and unstable compounds enamines/imines. In this method, the flavin adenine dinucleotide-dependent L- or D-amino acid oxidases generate an imino acid starting from a L- or D- amino acid, respectively. This reaction is coupled to the hydrolysis of the imino acid to the corresponding α-keto acid and ammonium ion catalyzed by a Rid enzyme. The spectrophotometric assay consists of measuring the decrease of the initial rate of formation of the semicarbazone, derived from the spontaneous reaction of the imino acid and semicarbazide, caused by the presence of the Rid enzyme. The set-up and testing of this method imply a preliminary characterization of the ability of the amino acid oxidase to release the imino acid required for the subsequent reactions. To this purpose, the activity of the L- or D-amino acid oxidases with different amino acids can be measured as production of hydrogen peroxide or formation of semicarbazone in parallel assays. The advantages and limitations of this assay of Rid activity are discussed.

A chiral GC-MS method for analysis of secondary amino acids after heptafluorobutyl chloroformate & methylamine derivatization.

L-amino acids (L-AAs) play different important roles in the physiology of all living organisms. Their chiral counterparts, D-amino acids (D-AAs) are increasingly being recognized as essential molecules in many biological systems. Secondary amino acids with cyclic structures, such as prolines, exhibit conformational rigidity and thus unique properties in the structural and protein folding. Despite their widespread occurrence, much less attention was paid to their chiral analysis, particularly when the minor, typically D-enantiomer, is present in low amounts in a complex biological matrix. In this paper, a cost-effective, chiral GC-MS method is described for capillary Chirasil-L-Val separation of nine cyclic secondary amino acid enantiomers with four-, five-, and six-membered rings, involving azetidine-2-carboxylic acid, pipecolic acid, nipecotic acid, proline, isomeric cis/trans 3-hydroxy, 4-hydroxyproline, and cis/trans-5-hydroxy-L-pipecolic acid in the excess of its enantiomeric antipode. The sample preparation involves in-situ derivatization with heptafluorobutyl chloroformate, simultaneous liquid-liquid micro-extraction into isooctane followed by amidation of the arising low-polar derivatives with methylamine, an evaporation step, re-dissolution, and final GC-MS analysis. The developed method was used for analyses of human biofluids, biologically active peptides containing chiral proline constituents, and collagen.

Effects of Hot-Pressure Extraction Time on Composition and Gelatin Properties of Chicken Bone Extracts.

Hot-pressure extraction was utilized in this study to extract proteins from chicken bones at 130 °C. The obtained extracts were further used to prepare gelatin gels. Results demonstrated that the extraction time can significantly affect the composition of the chicken bone extracts (P < 0.05). High-performance liquid chromatography (HPLC) analysis indicated that the protein fraction of molecular weight (MW) >30 KDa was only visible in the extracts collected between 40 and 60 min. The highest contents of hydroxyproline, imino acids, and hydrophobic amino acids were all achieved in the chicken bone extracts after 120 min of extraction, being 3.9, 7.7, and 16.0 mg/g, respectively. The prepared gelatin properties were evaluated in terms of viscosity, storage and loss modulus, stability, gel strength, and their microstructures. Results indicated that gelatins made from chicken bone extracts of 20, 40, and 60 min extraction had better properties compared to that of 90 and 120 min. Significant correlations were identified between gelatin's composition and properties (P < 0.05). The abundance of proteins with MW of <10 KDa and 10 to 30 KDa was found to be the predominant factor that can affect the gelatin's properties. This study illustrated a promising and natural way to obtain edible gelatins from chicken bones.

Comparison of physicochemical and functional properties of duck feet and bovine gelatins.

BACKGROUND: Previous studies have indicated that duck feet are a rich source of gelatin extractable from avian sources. In this study, the physicochemical and functional properties of avian gelatin extracted from duck feet (DFG) with acetic acid were compared with those of commercial bovine gelatin (BG). RESULTS: The yield of DFG obtained in this study was 7.01 ± 0.31%. High-performance liquid chromatography analysis indicated that the imino acid content was slightly lower for DFG compared with BG (P < 0.05). Differences in molecular size and amino acids between DFG and BG were also observed. The isoelectric points of DFG and BG were at pH 8 and 5 respectively, and the overall protein solubility of BG was higher than that of DFG. Gels prepared from BG exhibited higher bloom strength, viscosity and clarity and were darker in colour compared with DFG gels (P < 0.05). The gelling and melting points of BG were 21.8 and 29.47 °C respectively, while those of DFG were 20.5 and 27.8 °C respectively. BG exhibited slightly better emulsifying and foaming properties compared with DFG. CONCLUSION: Although some differences between DFG and BG were observed, the disparities were small, which indicates that DFG could be exploited commercially as an alternative source of gelatin. © 2016 Society of Chemical Industry.

Bone selective protective effect of a novel bone-seeking estrogen on trabecular bone in ovariectomized rats.

The drawbacks of estrogen restrict the clinical use of hormone replacement therapy, and it would be most helpful to explore new estrogenic substances that could prevent bone loss and be free from any adverse effects. We synthesized a new compound named bone-seeking estrogen (SE2) by combining 17ß-estradiol (E2) with iminodiacetic acid through the Mannich reaction. E2 and SE2 were labeled with isotope (3)H, and the tissue distribution tests of E2-(3)H and SE2-(3)H were analyzed by the radioactivity. The specific nuclear binding of E2 and SE2 in osteoblasts was measured. SE2 exhibited significantly greater affinity for bone but lower affinity for ovary and uterus than did E2, and SE2 maintained a high affinity for the estrogen receptor alpha similar to that of E2. SE2 administration did not induce uterine hypertrophy. Body weight increase was significantly suppressed by treatment with E2 but not by SE2 after ovariectomy (OVX). SE2 decreased bone turnover as E2 after OVX detected by serum biochemical markers. Bone histology and micro-CT analysis revealed that SE2 administration, similar to E2, could improve bone mass and trabecular architecture after OVX. Biomechanical analyses showed that SE2 treatment effectively increased mechanical properties after OVX. The results suggested that SE2 was effective in preventing OVX-induced bone loss and exhibited few side effects on body weight and uterine hypertrophy, which was beneficial in reducing the adverse effects caused by E2. SE2 may be a better choice than E2 for the prevention of postmenopausal osteoporosis.

State diagram of salmon (Salmo salar) gelatin films.

BACKGROUND: A state diagram presents different physical states of a biomaterial as a function of solid content and temperature. Despite their technological interest, little information is available on protein systems such as gelatin/water mixtures. The objective of this work was to develop state diagrams of salmon gelatin (SG) and bovine gelatin (BG) in order to determine maximal freeze concentration parameters (T'(g) , T'(m) and X(s') ) and to relate possible differences to their biochemical characteristics. RESULTS: Biochemical characterisation of SG showed lower molecular weight and iminoacid concentration compared with BG. Likewise, the glass transition temperature (T(g) ) was lower for SG at X(s) > 0.8, which was associated with its lower molecular weight. Unexpectedly, the depression of freezing temperature (T(f) ) was greater for SG at X(s) > 0.1, which was associated with its higher ash content. Isothermal annealing produced effective values of T'(g) ≈ - 52 °C, T'(m) ≈ - 46 °C and X'(s) ≈ 0.6 for both gelatins. Interestingly, the enthalpy change associated with T'(m) (ΔH T m) was significantly higher for SG than for BG after annealing, indicating a higher proportion of ice present at about - 50 °C. CONCLUSION: Maximal freeze concentration parameters were similar between the two gelatins, though differences in biochemical properties were evident. The results show that there are likely different ways of interaction of SG and BG with water.

Extraction and characterisation of pepsin-solubilised collagens from the skin of bigeye snapper (Priacanthus tayenus and Priacanthus macracanthus).

BACKGROUND: Fish collagen has been paid increasing attention as an alternative to the mammalian counterpart owing to the abundance of fish skin as a processing by-product. Generally, the low yield of collagen extracted using the typical acid solubilisation process has led to the use of mammalian pepsin as an aid for increasing the yield. Alternatively, fish pepsin, especially from tuna stomach, can be used for the extraction of pepsin-solubilised collagen (PSC). Therefore the objective of this study was to extract and characterise PSC from the skin of bigeye snapper, a fish widely used for surimi production in Thailand. RESULTS: PSCs from the skin of two species of bigeye snapper, Priacanthus tayenus and Priacanthus macracanthus, were extracted with the aid of tongol tuna (Thunnus tonggol) pepsin and porcine pepsin. PSCs from the skin of both species extracted using porcine pepsin had a higher content of beta-chain but a lower content of alpha-chains compared with those extracted using tuna pepsin. All PSCs contained glycine as the major amino acid and had an imino acid (proline and hydroxyproline) content of 189-193 residues per 1000 residues. Transition temperatures of PSCs were in the range 30.6-31.3 degrees C. Fourier transform infrared spectra revealed some differences in molecular order between PSCs extracted using porcine pepsin and tuna pepsin. Nevertheless, the triple-helical structure of PSCs was not affected by pepsin digestion. Zeta potential analysis indicated that PSCs from P. tayens and P. macracanthus possessed zero net charge at pH 7.15-7.46 and 5.97-6.44 respectively. CONCLUSION: Tongol tuna pepsin could be used as a replacement for mammalian pepsin in PSC extraction. However, a slight difference in PSC properties was found.

Dinitrophenyl derivatization of imino acids, spectral characteristics and HPLC analysis: application in urinary peptide-derived hydroxyproline and proline assay.

BACKGROUND: Assay of urinary imino acids, in particular peptide derived, is of immense utility in diagnosis of collagen-related disorders. The often-used methods for hydrolysis of urinary peptides need a long time and are cumbersome, hence the need for relatively simpler, but effective methods. METHODS: The method described, based on alkaline hydrolysis by autoclaving for 60 min followed by pre-column dinitrophenyl (DNP) derivatization and high-performance liquid chromatography (HPLC) analysis, demonstrates the complete hydrolysis and stability of urinary peptide derived imino acids. RESULTS: DNP derivatives of both imino acids had identical lambda max (380 nm) with molar epsilon of 28.224 x 10(3) and 17.036 x 10(3), respectively, for hydroxyproline (Hyp) and proline (Pro). HPLC run, extending up to 18 min, resolved major components of collagen products, namely Hyp, Hyl, Gly, Pro and Lys, with retention times of 6.5, 9.8, 10.5, 11.2 and 12.55 min, respectively. The assay method conformed to linear response for individual amino acid concentrations of 0.5-4.0 nmol per injection, with goodness of fit (r(2) value) 0.99 for both Hyp and Pro, and detection limit of 0.05-4.0 nmol of DNP derivatives. The recovery of Pro and Hyp, when spiked with urine prior to hydrolysis, were found to be 95% and 92%, respectively. CONCLUSION: Alkaline hydrolysis by autoclaving and DNP derivatization of imino acids followed by HPLC provides a method for the analysis of peptide-derived Hyp and Pro in urine. Hence, it is of utility to study collagen disorders.

Magnetic IDA-modified hydrophilic methacrylate-based polymer microspheres for IMAC protein separation.

Preparation of a new type of magnetic non-porous poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) microspheres with hydrophilic properties containing coupled iminodiacetic acid (IDA) is described. The prepared microspheres were used for the immobilization of Ni(II) or Fe(III) ions to show their application in protein binding studies. Human IgG was bound to magnetic Ni(II)-IDA-modified microspheres and conditions of its adsorption and elution were optimized. Non-specific binding of the protein to magnetic microspheres in the absence of Ni(II) ions was low. Fe(III) ions immobilized on magnetic IDA-modified microspheres were used for the specific binding of porcine pepsin, as a model phosphoprotein. The ability of phosphate buffer to release the adsorbed enzyme from the microspheres and a low adsorption of the dephosphorylated protein indicate the participation of phosphate groups in the pepsin interaction. The elaborated method represents a rapid technique that can be used not only for the separation of proteins but also for analytical purposes.

Investigation of mechanisms of oxidation of EDTA and NTA by permanganate at high pH.

Permanganate has been used for oxidation of nuclear wastes containing chelating agents such as ethylenediaminetetraacetic and nitrilotriacetic acids (EDTA and NTA) to improve separation of radionuclides and heavy metals from the wastes, butthe mechanisms of degradation of these and related organic ligands at high pHs have not been studied. EDTA, NTA, and the model compound ethylenediamine (EN) were found to be readily oxidized by permanganate at pH 12-14. The reduction of permangante was accompanied by formation of unstable manganate and dispersed MnO2 particles, which constituted the final product of permanganate reduction. The yields and speciation of EDTA, NTA, and EN breakdown products were affected by the pH and permanganate dose. Iminodiacetic acid (IDA), oxalate, formate, and ammonia were the predominant EDTA and NTA oxidation products. Mineralization of EDTA, NTA, and EN to CO2 was more significant at pH 12. At pH 14 formation of oxalate and deamination to NH3 were the most important reactions. IDA was released upon the oxidation of both EDTA and NTA, but EDTA oxidation yielded no ethylenediaminediacetic acid (EDDA). The speciation of the reaction products indicated that the ethylene group in EDTA was the preferred attack site in oxidations by alkaline permanganate.

HPLC method for determination of the purity of a mebrofenin-ligand for 99mTc-complexes.

A reversed-phase HPLC method has been developed for the estimation of purity and quantitative determination of mebrofenin UV detection at 205 nm was used. The stability of mebrofenin alone and in the presence of stannous chloride at 25 degrees C was studied. No decomposition product was found and there was no influence of SnCl2 on the stability of mebrofenin.

High-performance liquid chromatographic enantioseparation of unusual secondary amino acids on a D-penicillamine-based chiral ligand exchange column.

The application of a chiral ligand-exchange column (CLEC) for the direct high-performance liquid chromatographic enantioseparation of unusual secondary amino acids using D-penicillamine-Cu(II) complex as chiral selector is reported. The amino acids investigated were pyrrolidine-2-carboxylic acid, piperidine-2-carboxylic acid, piperazine-2-carboxylic acid, morpholine-3-carboxylic acid, and thiomorpholine-3-carboxylic acid analogs. Chromatographic results are given as the retention, separation, and resolution factors. The chromatographic conditions were varied to achieve optimal separation. The elution sequence of the enantiomers was determined and in most cases the S isomer eluted before R.

SclC is a member of a novel family of collagen-like proteins in Streptococcus equi subspecies equi that are recognised by antibodies against SclC.

Previously we have reported on a cell surface collagen-like protein, called SclC, from Streptococcus equi subspecies equi. In the present study we show that this protein is a member of a family of seven collagen-like proteins, called SclC-SclI in this subspecies. All proteins contain an N-terminal signal sequence, followed by a unique non-repetitive region called A, a highly repetitive collagen-like region (CL) consisting of Glycine-Xaa-Yaa-triplet repeats. Following the CL-region a C-terminal proline-rich putative wall spanning region (W) preceding an LPXTG-motif and a hydrophobic transmembrane region (M) are found, typical features of cell surface exposed proteins in Gram-positive bacteria. The nucleotide and amino acid sequences, were analysed to investigate the similarities between them, and recombinant proteins encoding different domains (A- and CL-regions) were expressed and purified. Although the novel collagen-like proteins display differences in amino acid sequences, affinity purified antibodies against SclC were found to cross react with the other members of the novel collagen-like proteins. Furthermore, in sera from horses previously diagnosed having strangles, antibodies against these proteins were detected suggesting that these proteins are expressed during the infection.

Alternative quality control for technetium-99m IDA complexes.

A quality control procedure for 99mTc-IDA complexes based on the use of C18 Sep-pak cartridges is developed and the validation of the procedure presented. C18 Sep-pak cartridges are pretreated by washing with 95% ethanol followed by 10(-3) N hydrochloric acid. A small amount of the 99mTc-IDA complex is applied, washed with 10(-3) N hydrochloric acid and eluted with 95% ethanol. The radiochemical purity values obtained for 99mTc-mebrofenin and 99mTc-disofenin using this Sep-pak procedure are comparable to those obtained using the standard two strip (ITLC-SG/100% methanol, ITLC-SA/20% saline) procedure.

Las proteínas / Proteins

La proteína es el principio constructor o plástico del organismo. La formación de la sangre, los músculos, el tejido cerebral, las hormonas o los anticuerpos dependen de un aporte adecuado de proteínas. Partiendo de esta premisa en el artículo se analiza sus aspectos bioquímicos, su valor biológico, cuáles son los aminoácidos esenciales y los limitantes, para acabar comentando algunos de los aspectos dietéticos más importantes de la alimentación vegetariana, relacionados con el aporte de proteínas (AU)

Quantification of duodenogastric reflux in patients with choledochoduodenostomy.

BACKGROUND: Frequently, patients present with symptoms after cholecystectomy (pain or discomfort in the upper part of the abdomen, postprandial fullness, bile vomiting, among others). Duodenogastric reflux has been associated with these symptoms in some patients. Therefore, this study was done to investigate this relationship. STUDY DESIGN: We evaluated duodenogastric reflux (DGR) in ten healthy patients, in ten patients who had asymptomatic simple cholecystectomy, in ten patients who had asymptomatic cholecystectomy with supraduodenal choledochoduodenostomy (CD), and in ten patients who had cholecystectomy plus CD followed by discomfort in the upper abdomen, postprandial fullness and bile vomiting, but no colicky pain or acute cholangitis. Duodenogastric reflux was quantified using continuous intravenous infusion of technetium-99m labeled hepatoiminodiacetic acid (99mTc-HIDA) and subsequently determining its concentration in gastric juice. RESULTS: All of the patients who underwent operation, whatever the technique used, had higher reflux rates than those in the control group (p < 0.001). Moreover, reflux rates were comparable in the patients who underwent simple cholecystectomy compared with patients in the asymptomatic cholecystectomy plus CD group. Conversely, when patients with cholecystectomy plus CD presented with discomfort in the upper part of the abdomen as well as bile vomiting, they had higher reflux rates than patients who underwent simple cholecystectomy (p < 0.001) and asymptomatic patients with associated CD (p < 0.001). CONCLUSIONS: Our results suggest that DGR must be involved in the genesis of these dyspeptic symptoms.

Duodenogastric reflux quantification in peptic ulcer surgery: comparison between different surgical techniques.

BACKGROUND: We quantified duodenogastric reflux with 6-hour continuous intravenous infusion of technetium 99m-labeled hepatoiminodiacetic acid (99mTc-HIDA) and subsequent quantification in gastric juice. METHODS: For this purpose, 50 patients were studied who had undergone surgery on the stomach with different surgical techniques: bilateral vagotomy plus Heineke-Mikulicz pyloroplasty, bilateral truncal vagotomy plus anterior pylorectomy, proximal gastric vagotomy, antrectomy and Billroth I reconstruction, and antrectomy and Billroth II reconstruction, comparing them with 10 healthy subjects used as a control group. We also studied the existing correlation between the rates of reflux determined by 99mTc-HIDA and those of total bile acids in gastric juice. RESULTS AND CONCLUSIONS: Patients who underwent gastric surgery had significantly greater quantities of duodenogastric reflux (p < 0.001) than had the control group. When the groups undergoing gastric surgery were compared, the patients who underwent resection showed higher reflux rates (p < 0.001) than did the patients who did not undergo resection. We found no differences among the groups of patients who did or did not undergo resection. We also found a highly significant correlation (p < 0.001) between the concentrations of 99mTc-HIDA and bile acids in gastric juice.

Efficient enantioselective separation and determination of trace impurities in secondary amino acids (i.e., imino acids).

An R-(-)-1-(1-naphthyl)ethyl carbamoylated-beta-cyclodextrin bonded phase in conjunction with a nonaqueous polar mobile phase was used for the highly selective enantioseparation of a number of secondary amino acids after their pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC). Under the conditions employed, the FMOC reagent served to "lock" the imino acid into their existing conformation thereby preventing the possibility of racemization. Furthermore, it served to increase the sensitivity to the point that trace level enantiomeric impurities were easily detected. Compared with separations that use traditional reversed-phase solvents, this method showed several advantages: higher selectivity towards the imino acid enantiomers investigated, shorter analysis times, faster equilibration of the column, more stable baseline and more sensitive fluorescence detection. The detection limits for FMOC derivatives of proline, trans-4-hydroxyproline, cis-4-hydroxyproline, pyroglutamic acid, 3,4-dehydroproline, thiaproline, penicillamine acetone adduct and pipecolic acid are in the low femtomole range. The method was used for evaluation of enantioselectivity of a number of "optically pure" commercial imino acid standards. Enantiomeric impurities as low as 0.0001% (1 ppm) can be determined in some cases. High precision determination of trace levels of D-imino acids in the presence of large amounts of corresponding (opposite) L enantiomer at 1, 0.1, 0.01% and below are demonstrated.