Proteins and protein components for sportspeople: quality control of dietary supplements.

Quality control of protein supplements intended for a large audience of consumers such as sportspeople is particularly important. A case study on quality control of dietary supplements containing protein and protein components is presented. The objective of the study was to evaluate the conformity of the quantities of amino acids, essential and branched-chain amino acids, declared on the label through measurements with chromatographic analytical tools. 16 sportspeople supplements from different European countries were tested. Analysis of concentrated whey protein highlighted some differences between the label and what was experimentally determined; in these samples some amino acids (6 amino acids out of 19) exceeded the maximum tolerance (>20%) regulated by the European Commission. To a lesser extent, analysis of the other classes revealed amino acid concentrations that exceeded the maximum analytical tolerance percentage. As regards the essential and branched amino acid supplements, it was seen that the declared quantity conforms with that determined experimentally.

Automated sample preparation with 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate and iodoacetamide derivatization reagents for enantioselective liquid chromatography tandem mass spectrometry amino acid analysis.

Enantioselective amino acid analysis is gaining increasing importance in pharmaceutical, biomedical and food sciences. While there are many methods available for enantiomer separation of amino acids, the simultaneous analysis of all chiral proteinogenic amino acids by a single method with one column and a single condition is still challenging. Herein, we report an enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS) assay using Chiralpak QN-AX as chiral column. With 6-aminoquinolyl-N-hydrosysuccinimidyl carbamate (AQC) as derivatization reagent, efficient enantioselective separation of D- and L-amino acids using HPLC has become possible. Thiol-containing amino acids like Cys are alkylated prior to AQC-labelling. A protocol for automated sample preparation including both derivatization step and calibrator preparation is presented. For compensating matrix effects, u-13C15N-labelled internal standards (IS) were employed. The method was validated and applied to the enantioselective analysis of amino acids in a bacterial fermentation broth.

Development of a novel single-chain l-glutamate oxidase from Streptomyces sp. X-119-6 by inserting flexible linkers.

L-glutamate oxidase (LGOX, EC: 1.4.3.11) is an oxidoreductase that catalyzes L-glutamate deamination. LGOX from Streptomyces sp. X-119-6 is used widely for L-glutamate quantification in research and industrial applications. This enzyme encoded as a single precursor chain that undergoes post-translational cleavage to four fragments by an endogenous protease to become highly active. Efficient preparation of active LGOX by heterologous expression without proteolysis process should be indispensable for wide application of this enzyme. Thus, developing an LGOX that requires no protease treatment should expand the potential applications of recombinant LGOX. In this report, we succeeded in obtaining an active single-chain LGOX by connecting the four fragments of the mature form with insertion of flexible linkers. The most active single-chain mutant showed the similar activity to that of the mature form from Streptomyces sp. X-119-6. The structure of this mutant was determined at 2.9 Å resolution by X-ray crystallography. It was revealed that this single-stranded mutant had the similar conformation to that of mature form. This single-chain LGOX can be produced efficiently and should expand LGOX applications.

In vitro digestion characteristics of cereal protein concentrates as assessed using a pepsin-pancreatin digestion model.

An alkaline extraction method has been used in many studies to extract total protein from cereal samples. Wheat bran protein concentrate (WBPC), oat bran protein concentrate (OBPC), and barley protein concentrate (BPC) were prepared by alkaline extraction and isoelectric precipitation to study their functional and nutritional properties. The three protein concentrates were hydrolysed by an in vitro pepsin-pancreatin digestion model. Their digestibility (%) and degree of hydrolysis (DH%) were evaluated, and SDS-PAGE electrophoresis was used to illustrate the protein and peptides patterns. The change of the particle sizes and the release of the essential amino acids was followed during the digestion process. The in vitro digestibility of WBPC, OBPC and BPC was 87.4%, 96.1% and 76.9%, respectively. The DH% of protein concentrates were between 50 and 60%. The change of the particle size distribution values Dv(50) was assumed to be related to protein aggregations during the digestion. The protein fractions were identified and the degradation during the digestion and were analysed by SDS-PAGE; the gels of WBPC and OBPC digestion showed virtually complete degradation whereas the intensive bands of undigested protein were presented for BPC. The generation of the free amino acids and short chain peptides were significantly higher at the end of the intestinal digestion compared to the stages of before and after gastric digestion. Higher content of the deficient amino acids such as lysine and threonine were found comparing to the level of deficient amino acids in cereal grains but does not meet the daily recommended intake.

Automatic switching valve system to minimize variation of liquid chromatography-tandem mass spectrometry-based chiral amino acid profiling.

Although d-amino acids are less prevalent in nature, they have been detected in mammals (including humans) and it is widely accepted that they might play important physiological roles. While an analytical method for chiral amino acid profiling is strongly required, it has not been well-established because of the difficulties associated with analysis. A high-sensitivity and high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical method was recently reported by our group for chiral amino acids; however, it lacked sufficient repeatability for several d-amino acids. Thus, the aim of this research was to reduce the experimental variation of chiral amino acid analysis. By installing an automatic switching valve system in LC-MS/MS, it was possible to reduce the relative standard deviations of d-amino acid ratios (d/(d+l)) in rat urine obtained from three technical replicates. The results indicated that the automatic switching valve system was effective in minimizing the variation of d-amino acid ratios, and could be applied for profiling d-amino acids because of its high repeatability.

Advances in research on mass spectrometry based chiral amino acid analysis for quality control of racemic peptide impurities / 药学学报

Chiral amino acid analysis is a sensitive, efficient and economical method for controlling racemic peptide impurities, especially for synthetic polypeptide drugs with complex composition of amino acids. Unexpected amino acid enantiomers in racemic peptides can be measured by chiral amino acid analysis coupled with mass spectrometry. The position of amino acid isomerization in the peptide segment can be accurately mapped by mass spectrometry, which lays a solid foundation for screening of racemic peptide impurities and rapid identification or quantification of trace racemic peptide impurities. Combination of the two techniques is vital for quality control of the synthetic polypeptide drugs and for research of polypeptide drugs based on chemical synthesis. The strategies of peptide hydrolysis have been summarized in this review. The latest chiral amino acid analysis based on mass spectrometry is briefly reviewed. Based on our knowledge, we have pointed to the direction of research and control of racemic peptide impurities in synthetic polypeptide drugs.

CE-MS for the Analysis of Amino Acids.

Amino acids play an important role in clinical analysis. Capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) has proven to possess several characteristics that make it a powerful and useful tool for the analysis of amino acids in clinical studies. Here we present a method for the separation and quantitative analysis of 27 amino acids in urine based on CE-ESI-MS. The method presents an improved resolution between the isomers Leu, Ile, and aIle, in comparison to other CE-ESI-MS methods in the literature. This method is fast, selective, and simple and has improved sensitivity by applying a pH-mediated stacking strategy, showing that it can be successfully used for amino acid analysis and probably for other small cationic metabolites.

Analysis of amino acids and acyl carnitine profiles in low birth weight, preterm, and small for gestational age neonates.

OBJECTIVE: To analyze the amino acids (AA) and acyl carnitine (AC) profiles in dry blood spot (DBS) specimens of low birth weight (LBW), preterm birth (PTB), and small for gestational age (SGA), and to compare the concentration difference of AA and AC with those without above. METHODS: This is a retrospectively study. Eight thousand nine hundred and seventy-nine uncomplicated pregnant newborns were enrolled into the study. DBS were collected on the third day of life, and concentrations of 11 types of AA, free carnitine and 30 types of AC were detected by using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS). Shapiro-Wilk test and Kruskal-Wallis rank test were applied in statistical analysis. RESULTS: Concentrations of most AA and AC in infants born in SGA were significantly higher than those in non-SGA group, while lower in LBW and PTB groups than those in non-LBW and non-PTB groups (p < 0.05). CONCLUSIONS: The difference of concentration of AA and AC in the subgroups suggested there may be a dysutilization of AA and AC in SGA, but an inborn insufficient of AA and AC in LBW and PTB neonates.

Digital microfluidics platform for interfacing solid-liquid extraction column with portable capillary electropherograph for analysis of soil amino acids.

In this work, the concept of a field-portable analyzer is proposed that operates with milliliter amounts of solvents and samples. The need to develop such an analyzer is not only driven by specific extraterrestrial analysis but also, for example, by forensics applications where the amount of liquid that can be taken to the field is severely limited. The prototype of the proposed analyzer consists of a solid-liquid extractor, the output of which is connected to the micropump, which delivers droplets of extracts to digital microfluidic platform (DMFP). In this way, world-to-chip interfacing is established. Further, the sample droplets are transported to CE capillary inlet port, separated and detected via a contactless conductivity detector. Working buffers and other solvents needed to perform CE analysis are also delivered as droplets to the DMFP and transported through the CE capillary. The performance of the analyzer is demonstrated by analysis of amino acids in sand matrices. The recovery of the spiked amino acids from the inert sand sample was from 34 to 51% with analysis LOD from 0.2 to 0.6 ppm and migration time RSD from 0.2 to 6.0%.

Comparison of cake compositions, pepsin digestibility and amino acids concentration of proteins isolated from black mustard and yellow mustard cakes.

As a byproduct of oil production, black and yellow mustard cakes protein are considered as potential source of plant protein for feed applications to poultry, fish and swine industries. The protein contents in black and yellow mustard cakes were 38.17% and 28.80% and their pepsin digestibility was 80.33% and 77.43%, respectively. The proteins were extracted at different pH and maximum proteins (89.13% of 38.17% and 87.76% of 28.80% respectively) isolated from black and yellow mustard cakes at pH 12. The purity of isolated proteins of black and yellow mustard cakes was 89.83% and 91.12% respectively and their pepsin digestibility was 89.67% and 90.17% respectively which assigned the absence of antinutritional compounds. It was found that essential amino acids isoleucine, lysine, methionine, threonine and tryptophan and non essential amino acids arginine and tyrosine were present in greater concentration in black mustard cake protein whereas other amino acids were higher in yellow mustard cake protein.

Study on Fingerprint of Amino Acids in Honey by High Performance Liquid Chromatography / 广州中医药大学学报

Objective To establish a method for fingerprint analysis of amino acids from honey by high performance liquid chromatography ( HPLC). Methods Amino acids of honey were concentrated by 732 cation exchange resin, and then were treated by pre-column derivatization with phenyl isothiocyanate, with praline as control peak. The chromatography was performed on a Waters Symmetry C18 ( 250 mm × 4.6 mm × 5 μm) column, with acetonitrile ∶ water (4∶1) as mobile phase A and 30 mmol/L sodium acetate ∶ acetonitrile (355∶15, acetic acid adjusting pH value to be 6.5) as mobile phase B by gradient elution. The detection wave length was set at 254 nm. The flow rate was 1.0 mL/min. The column temperature was 40℃, and the injection volume was 5μL. Results Sixteen common peaks were shown in the fingerprint of 15 batches of honey samples. The similarity for 15 batches of honey samples was in the range of 0.910 ~ 0.996 . Conclusion The fingerprint detection method is simple, practical, reproducible and specific, and can provide certain reference for quality control of honey.

Anti-diabetic and antihypertensive activities of two flaxseed protein hydrolysate fractions revealed following their simultaneous separation by electrodialysis with ultrafiltration membranes.

Flaxseed protein hydrolysate has been fractionated by electrodialysis with two ultrafiltration membranes (20 and 50 kDa) stacked in the system for the recovery of two specific cationic peptide fractions (KCl-F1 and KCl-F2). After 360 min of treatment, peptide migration increased as a function of time in KCl compartments. Moreover, the use of two different ultrafiltration membrane allowed concentration of the 300-400 and 400-500 Da molecular weight range peptides in the KCl-F1 and KCl-F2 fractions, respectively, compared to the initial hydrolysate. After mass spectrometry analysis, higher amounts of low molecular weight peptides were recovered in the KCl-F2 compartment while relatively higher molecular weight peptides were more detected in the KCl-F1 compartment. Amino acid analysis showed that His, Lys and Arg were especially concentrated in the KCl compartments. Finally, glucose-transport assay demonstrated that the KCl-F2 fraction increased glucose uptake while oral administration of KCl-F1 and final FPH decreased systolic blood pressure.

Comparative Analysis of Amino Acids of Lignum Santali Albi from Different Habitats / 广州中医药大学学报

【Objective】To compare the fingerprint spectrum of amino acids(AA)of Lignum Santali Albi(LSA)from Indonesia,India and Australia.【Methods】Free AA and hydrolytic AA of LSA from Indonesia,India and Australia were analyzed by amino acid automatic analyzer.The detection wavelength was 570nm and 440nm.【Results】There were obvious differences among the free AA from three kinds of LSA.2S,4S-4-hydroxyproline(Hyp)was detected in Indonesian LSA in 11.77min(?=440nm)with the content of 1.431?7%,and became proline after hydrolysis.The peak of Hyp was not obvious or even undetectable in Indian LAS and Australian LSA.【Conclusion】There exist obvious differences among free AA from three kinds of LSA and 2S,4S-4-Hyp can be used as the specific compounds for the identification of LSA from Indonesia,India and Australia.

The variation of four amino acids release of the retina in acute ocular hypertension in rabbits / 中华眼底病杂志

Objective To monitor the release of amino acids of the whole retina during and after experimental glaucoma by increasing the intraocular pressure (IOP). Methods Experimental glaucoma was induced in one of the two eyes of rabbits by increasing IOP at 120 mm Hg for 45 min under infusion of saline in anterior chamber;then the pressure was released and the needle inserted into the anterior chamber was removed,this state was maintained for another 45 min.Every 15 min during the experiment 5 rabbits were killed and experimental eyes were enucleated.Aliquots (20 ?l) of the retinal extracts (see below) were mixed with ophthaldialdehyde reagent and analysed for amino acid content by the HPLC method of Wangwei, using a 150 mm?4.6 mm,5 ?m C18 column. Results A large increase in the release of glutamate,but not of the other three amino acids monitored,occurred during initial experimental ocular hypertension.It reached peak value of (111.73?17.46) 10 -5 mmol/g at 15 min of hypertension.15 min after release of intraocular pressure,again,immediately large and specific increase in the concentration of glutamate was reached to (102.96?51.91) 10 -5 mmol/g.In eyes subjected to paracentesis of anterior chamber,no difference was found between experimental eyes and controls. Conclusion These results suggest that glutamate is triggered by increasing the IOP,and it releases not only during the period of experimental ocular hypertension,but also afterwards.

Analysis of amino acid constituents of gallstones.

AIM: To seek drugs that will efficaciously dissolve bilirubin, glycoprotein and black stones and that will represent improved lithotriptic agents to resolve cholesterol stones, and to study the amino acid constituents of gallstones. METHODS: According to characteristics determined by infrared spectroscopy and to the contents of bilirubin determined by semi-quantitative chemical analysis, 30 of 148 cases of gallstones were selected and divided into 5 groups. Amino acids of the 30 cases were detected by high-speed chromatography. RESULTS: The quantity of amino acids was highest in black stones (226.9 mg/g) and lowest in pure cholesterol stones (1.4 mg/g). In the 5 groups of gallstones, the quantity of amino acids followed the hierarchy of black stone > mixed bilirubin stone and glucoprotein stone > mixed cholesterol stone > pure cholesterol stone. The proportions were: 95.95:29.02 and 28.05:5.78:1. Aliphatic amino acids accounted for approximately 50% of the total amino acids in the gallstones, with glycine accounting for 15.3% of the total amount of the 17 kinds of amino acids. CONCLUSION: For mixed stones, the higher level of bilirubin, the higher content of amino acids. Acidic amino acids were relatively higher in bilirubin stones than in cholesterol stones.