Intensive determination of storage condition effects on human plasma metabolomics

Introduction

Human plasma metabolomics offer powerful tools for understanding disease mechanisms and identifying clinical biomarkers for diagnosis, efficacy prediction and patient stratification. Although storage conditions can affect the reliability of data from metabolites, strict control of these conditions remains challenging, particularly when clinical samples are included from multiple centers. Therefore, it is necessary to consider stability profiles of each analyte.

Objectives

The purpose of this study was to extract unstable metabolites from vast metabolome data and identify factors that cause instability.

Method

Plasma samples were obtained from five healthy volunteers, were stored under ten different conditions of time and temperature and were quantified using leading-edge metabolomics. Instability was evaluated by comparing quantitation values under each storage condition with those obtained after ?80 °C storage.

Result

Stability profiling of the 992 metabolites showed time- and temperature-dependent increases in numbers of significantly changed metabolites. This large volume of data enabled comparisons of unstable metabolites with their related molecules and allowed identification of causative factors, including compound-specific enzymatic activity in plasma and chemical reactivity. Furthermore, these analyses indicated extreme instability of 1-docosahexaenoylglycerol, 1-arachidonoylglycerophosphate, cystine, cysteine and N⁶-methyladenosine.

Conclusion

A large volume of data regarding storage stability was obtained. These data are a contribution to the discovery of biomarker candidates without misselection based on unreliable values and to the establishment of suitable handling procedures for targeted biomarker quantification.

     

Isolation and Characterisation of Insulin-Releasing Compounds from <Emphasis Type="Italic">Pseudechis australis</Emphasis> and <Emphasis Type="Italic">Pseudechis butleri</Emphasis> Venom

Crude venom from two elapid snakes Pseudechis australis and Pseudechis butleri was fractionated by gel filtration chromatography and selected fractions screened for in vitro insulin-releasing activity using clonal pancreatic BRIN-BD11 cells. Following acute 20-min incubation at 5.6 mM glucose, 9 fractions exhibited significant (P < 0.001) insulin-releasing activity. Structural characterisation of active fractions was achieved primarily using MALDI–TOF MS and N-terminal Edman degradation sequencing. The partial N-terminal sequences are reported for a total of 7 venom components. Their homology to existing sequences as determined using BLAST searching uncovered the main insulin-releasing families as being phospholipases A₂ and short α-neurotoxins. A number of sequences are reported for the first time from P. butleri venom which is much less studied than the related P. australis.     

Effect of Cadmium Ion on alpha-Glucosidase: An Inhibition Kinetics and Molecular Dynamics Simulation Integration Study

α-Glucosidase is a critical metabolic enzyme that produces glucose molecules by catalyzing carbohydrates. The aim of this study is to elucidate biological toxicity of Cd²⁺ based on α-glucosidase activity and conformational changes. We studied Cd²⁺-mediated inactivation as well as conformational modulation of α-glucosidase by using kinetics coupled with simulation of molecular dynamics. The enzyme was significantly inactivated by Cd²⁺ in a reversibly binding behavior, and Cd²⁺ binding induced a non-competitive type of inhibition reaction (the K _i was calculated as 0.3863 ± 0.033 mM). Cd²⁺ also modulated regional denaturation of the active site pocket as well as overall partial tertiary structural change. In computational simulations using molecular dynamics, simulated introduction of Cd²⁺ induced in a depletion of secondary structure by docking Cd²⁺ near the saccharides degradation at the active site, suggesting that Cd²⁺ modulating enzyme denaturation. The present study elucidated that the binding of Cd²⁺ triggers conformational changes of α-glucosidase as well as inactivates catalytic function, and thus suggests an explanation of the deleterious effects of Cd²⁺ on α-glucosidase.     

Can Carnosine Prevent the Aging-Induced Changes of Blood Platelet and Brain Regional Monoamine Oxidase-A mRNA in Relation to its Activity?

It is well known that the monoamine oxidase (MAO) activity deregulates during aging along with anti-oxidant activity. Carnosine (β-Ala-l-His) is an endogenous dipeptide biomolecule, having both anti-oxidant and anti-glycating properties. The present study deals with the effect of carnosine on aging-induced changes in MAO-A mRNA expression of brain regions and blood platelets in relation to their MAO-A activity. Results showed that aging significantly and characteristically increased the brain regional MAO-A mRNA whereas, in blood platelets it was significantly reduced with an increase in blood platelet counts. Carnosine attenuated both aging-induced (i) increase in brain regional MAO-A mRNA expression and blood platelet count, (ii) decrease in blood platelet MAO-A mRNA expression and its (platelet MAO-A) activity without affecting the young rats’ brain regions and platelet. The present results thus suggest that carnosine attenuated and restored the aging-induced (a) increase of platelet count and (b) changes in brain regional and blood platelet MAO-A mRNA expression and (c) decrease in platelet MAO-A activity, towards their respective basal level that were observed in young rats.     

Identification of Miscellaneous Peptides from the Skin Secretion of the European Edible Frog, <Emphasis Type="Italic">Pelophylax kl. Esculentus</Emphasis>

The chemical compounds synthesised and secreted from the dermal glands of amphibian have diverse bioactivities that play key roles in the hosts’ innate immune system and in causing diverse pharmacological effects in predators that may ingest the defensive skin secretions. As new biotechnological methods have developed, increasing numbers of novel peptides with novel activities have been discovered from this source of natural compounds. In this study, a number of defensive skin secretion peptide sequences were obtained from the European edible frog, P. kl. esculentus, using a ‘shotgun’ cloning technique developed previously within our laboratory. Some of these sequences have been previously reported but had either obtained from other species or were isolated using different methods. Two new skin peptides are described here for the first time. Esculentin-2c and Brevinin-2Tbe belong to the Esculentin-2 and Brevinin-2 families, respectively, and both are very similar to their respective analogues but with a few amino acid differences. Further, [Asn-3, Lys-6, Phe-13] 3-14-bombesin isolated previously from the skin of the marsh frog, Rana ridibunda, was identified here in the skin of P. kl. esculentus. Studies such as this can provide a rapid elucidation of peptide and corresponding DNA sequences from unstudied species of frogs and can rapidly provide a basis for related scientific studies such as those involved in systematic or the evolution of a large diverse gene family and usage by biomedical researchers as a source of potential novel drug leads or pharmacological agents.     

Oxytocin Sustained Release Using Natural Rubber Latex Membranes

The demand for biomaterials with properties that provide sustained release of substances with pharmacological interest is constant. One candidate for applications in this area is the Natural Rubber Latex (NRL) extracted from the rubber tree Hevea brasiliensis. Recent studies indicate the NRL as a matrix for sustained release, showing promising results for biomedical applications such as: can stimulate natural angiogenesis and is capable of adhering cells on its surface, promoting the replacement and regeneration of tissue. So, the NRL is an excellent candidate to propitiate the sustained release of peptides of pharmacological interest such as oxytocin, a hormonal peptide which has the function to promote uterine muscle contractions and reduce bleeding during childbirth, and stimulate the release of breast milk. Results demonstrated that 90 μg mL^?1 (45 %) of the incorporated peptide in Natural Rubber Latex Biomedical (NRLb) functionalized membranes was released at 10 h in phosphate-buffered saline (PBS) solution. Swelling kinetics assay showed that the NRLb membranes are able to absorb over a period of 16 h up to 1.08 grams of water per grams of membrane. Scanning electron microscopy showed that the peptide was adsorbed on the surface and within NRLb membrane. Fourier transform infrared and Derivative Thermogravimetric analysis indicated that oxytocin did not interacted chemically with the membrane. Furthermore, hemolysis of erythrocytes, quantified spectrophotometrically using materials (Oxytocin, NRLb, and NRLb + Oxytocin) showed no hemolytic effects up to 100 μg mL^?1 (compounds and mixtures), indicating no detectable disturbance of the red blood cell membranes. Based on these results it was possible to conclude that the NRLb has shown effectiveness as a model in the release of peptides with pharmacological interest.     

Novel Dipeptidyl Peptidase IV Inhibitory and Antioxidant Peptides Derived from Human Gastrointestinal Endogenous Proteins

Human gastrointestinal endogenous proteins (GEP) include the proteins mucins, serum albumin, digestive enzymes, and proteins from sloughed epithelial and microbial-cells. GEP play a vital role in the digestion of food, but are also simultaneously digested by proteases and peptidases of the gastrointestinal tract (GIT). Recent studies suggest that during gastrointestinal digestion, similar to dietary proteins, GEP may also give rise to bioactive peptides. In the present study, the protein sequences of 11 representative GEP were subjected to simulated in silico GIT (SIGIT) digestion. Following SIGIT digestion, 19 novel GEP-derived peptide sequences were selected using quantitative structure activity relationship rules for chemical synthesis. The peptides were then tested for their in vitro dipeptidyl peptidase IV (DPP-IV) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition, and for their ferric reducing antioxidant power (FRAP). Two novel DPP-IV inhibitory peptides with the amino acid sequences RPCF (IC₅₀ = 800.51 ± 49.00 µM) and MIM (IC₅₀ = 1056.78 ± 61.11 µM), and five novel antioxidant peptides CCK, RPCF, CRPK, QQCP and DCR were identified. The results of this study indicate that GEP are a significant source of bioactive peptides with potential novel bioactive peptide fragments within their sequences.     

Enhancing the Yield of Active Recombinant Chitobiase by Physico-Chemical and In Vitro Refolding Studies

Chitobiase (CHB) is an important enzyme for the production of N-acetyl-d-glucosamine from the chitin biopolymer in the series of chitinolytic enzymes. Majority of over-expressed CHB (58 %) in E. coli expression system led to formation of inclusion bodies. The production and soluble yield of active CHB was enhanced by co-expression with GroEL/ES chaperonin, optimizing culture conditions and solubilization followed by refolding of remaining inactive chitobiase present in the form of inclusion bodies. The growth of recombinant E. coli produced 42 % CHB in soluble form and the rest (~58 %) as inclusion bodies. The percentage of active CHB was enhanced to 71 % by co-expression with GroEL/ES chaperonin system and optimizing culture conditions (37 °C, 200 rpm, IPTG—0.5 mM, l-arabinose—13.2 mM). Of the remaining inactive CHB present in inclusion bodies, 37 % could be recovered in active form using pulsatile dilution method involving denaturants (2 M urea, pH 12.5) and protein refolding studies (1.0 M l-arginine, 5 % glycerol). Using combinatorial approach, 80 % of the total CHB expressed, could be recovered from cells grown in one litre of LB medium is a step forward in replacing hazardous chemical technology by biotechnological process for the production of NAG from chitinous waste.