Protein Precipitation by Metal Hydroxides as a Convenient and Alternative Sample Preparation Procedure for Bioanalysis.

Protein precipitation is widely used for sample preparation ahead of liquid chromatography. This step is required to analyze small molecules without the interference of proteins contained in the matrix. Organic solvents and acidic chemicals are the two most popular reagents used for this scope.

Exploring Secondary Amine Carnosine Derivatives: Design, Synthesis, and Properties.

Carnosine is a naturally occurring dipeptide that has been advocated by some authors as an interesting scaffold for the development of potential therapeutic agents in view of the positive outcomes of its supplementation in animal models of human diseases. Its mode of action seems to depend on the quenching of toxic electrophiles, such as 4-hydroxynonenal (HNE). However, carnosine's bioavailability in humans is lower than that in other mammals.

State of the Art in the Development of Human Serum Carnosinase Inhibitors.

Human serum carnosinase is an enzyme that operates the preferential hydrolysis of dipeptides with a C-terminus histidine. Only higher primates excrete such an enzyme in serum and cerebrospinal fluid. In humans, the serum hydrolytic rate has high interindividual variability owing to gene polymorphism, although age, gender, diet, and also diseases and surgical interventions can modify serum activity.

A Novel Chromatographic Method to Assess the Binding Ability towards Dicarbonyls.

Human exposure to dicarbonyls occurs via ingestion (e.g., food), inhalation (e.

Volumetric Absorptive Microsampling (VAMS) for Targeted LC-MS/MS Determination of Tryptophan-Related Biomarkers.

L-Tryptophan (TRP) metabolites and related biomarkers play crucial roles in physiological functions, and their imbalances are implicated in central nervous system pathologies and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease, schizophrenia and depression. The measurement of TRP metabolites and related biomarkers possesses great potential to elucidate the disease mechanisms, aid preclinical drug development, highlight potential therapeutic targets and evaluate the outcomes of therapeutic interventions. An effective, straightforward, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of 24 TRP-related compounds in miniaturised murine whole blood samples.

Chromatographic measurement of 3-hydroxyanthranilate 3,4-dioxygenase activity reveals that edaravone can mitigate the formation of quinolinic acid through a direct enzyme inhibition.

Herein it is reported the development and application of two chromatographic assays for the measurement of the activity of 3-Hydroxyanthranilate-3,4-dioxygenase (3HAO). Such an enzyme converts 3-Hydroxyanthranilic acid (3HAA) to 2-amino-3-carboxymuconic semialdehyde (ACMS), which undergo a spontaneous, non-enzymatic cyclization to produce quinolinic acid (QUIN). The enzyme activity was measured by quantitation of the substrate consumption over time either with spectrophotometric (UV) or mass spectrometric (MS) detection upon reversed-phase chromatographic separation.

The ergogenic effect of acute carnosine and anserine supplementation: dosing, timing, and underlying mechanism.

Background: Recent studies suggest that acute-combined carnosine and anserine supplementation has the potential to improve the performance of certain cycling protocols. Yet, data on optimal dose, timing of ingestion, effective exercise range, and mode of action are lacking. Three studies were conducted to establish dosing and timing guidelines concerning carnosine and anserine intake and to unravel the mechanism underlying the ergogenic effects.

Synthesis and characterization of C labeled carnosine derivatives for isotope dilution mass spectrometry measurements in biological matrices.

Due to the physiological properties of l-carnosine (l-1), supplementation of this dipeptide has both a nutritional ergogenic application and a therapeutic potential for the treatment of numerous diseases in which ischemic or oxidative stress are involved. Quantitation of carnosine and its analogs in biological matrices results to be crucial for these applications and HPLC-MS procedures with isotope-labeled internal standards are the state-of-the-art approach for this analytical need. The use of these standards allows to account for variations during the sample preparation process, between-sample matrix effects, and variations in instrument performance over analysis time.

Characterization of Catecholaldehyde Adducts with Carnosine and l-Cysteine Reveals Their Potential as Biomarkers of Catecholaminergic Stress.

Monoamine oxidase (MAO) catalyzes the oxidative deamination of dopamine and norepinephrine to produce 3,4-dihydroxyphenylacetaldehyde (DOPAL) and 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL), respectively. Both of these aldehydes are potently cytotoxic and have been implicated in pathogenesis of neurodegenerative and cardiometabolic disorders. Previous work has demonstrated that both the catechol and aldehyde moieties of DOPAL are reactive and cytotoxic via their propensity to cause macromolecular cross-linking.

Acute preexercise supplementation of combined carnosine and anserine enhances initial maximal power of Wingate tests in humans.

Classic in vitro experiments (Severin's phenomenon) demonstrated that acute carnosine supplementation may potentiate muscle contractility. However, upon oral ingestion, carnosine is readily degraded in human plasma by the highly active serum carnosinase-1 (CN1). We developed a novel strategy to circumvent CN1 by preexercise ingestion of combined carnosine (CARN) and anserine (ANS), the methylated analog with similar biochemical properties but more resistant to CN1.

Understanding the antioxidant and carbonyl sequestering activity of carnosine: direct and indirect mechanisms.

Carnosine is an endogenous dipeptide whose oral administration has been found to prevent several oxidative based diseases including lung disease, type 2 diabetes and its micro and macrovascular complications, cardiovascular disorders, neurodegenerative and kidney disease. While it is generally accepted that the beneficial effects of carnosine are due to its antioxidant, anti-advanced glycation end product (AGE) and -advanced lipoxidation end product (ALE) and anti-inflammatory properties, the molecular mechanisms explaining such effects have not yet been clearly defined. Studies indicate that carnosine acts by a direct antioxidant mechanism and by sequestering reactive carbonyls (RCS), the byproducts of lipid and glucose oxidation, thus inhibiting AGE and ALE which are the reaction products of RCS with proteins.

Towards the Inhibition of Protein-Protein Interactions (PPIs) in STAT3: Insights into a New Class of Benzothiadiazole Derivatives.

Signal transducer and activator of transcription 3 (STAT3) is a validated anticancer target due to the relationship between its constitutive activation and malignant tumors. Through a virtual screening approach on the STAT3-SH2 domain, 5,6-dimethyl-1,3-2,1,3-benzothiadiazole-2,2-dioxide () was identified as a potential STAT3 inhibitor. Some benzothiadiazole derivatives were synthesized by employing a versatile methodology, and they were tested by an AlphaScreen-based assay.

Development of a direct LC-ESI-MS method for the measurement of human serum carnosinase activity.

Carnosine (β-alanyl-L-histidine) is a natural peptide that have been described as a potential pharmacological agent owing to some positive outcomes from several pharmacological tests in animal models of human diseases. However, carnosine has limited activity in humans since the peptide upon absorption is rapidly hydrolyzed in the serum by the enzyme carnosinase (i.e.

Two novel fish paralogs provide insights into the Rid family of imine deaminases active in pre-empting enamine/imine metabolic damage.

Reactive Intermediate Deaminase (Rid) protein superfamily includes eight families among which the RidA is conserved in all domains of life. RidA proteins accelerate the deamination of the reactive 2-aminoacrylate (2AA), an enamine produced by some pyridoxal phosphate (PLP)-dependent enzymes. 2AA accumulation inhibits target enzymes with a detrimental impact on fitness.

In Vitro Aging of Human Skin Fibroblasts: Age-Dependent Changes in 4-Hydroxynonenal Metabolism.

Evidence suggests that the increased production of free radicals and reactive oxygen species lead to cellular aging. One of the consequences is lipid peroxidation generating reactive aldehydic products, such as 4-hydroxynonenal (HNE) that modify proteins and form adducts with DNA bases. To prevent damage by HNE, it is metabolized.

Tuning PFKFB3 Bisphosphatase Activity Through Allosteric Interference.

The human inducible phospho-fructokinase bisphosphatase isoform 3, PFKFB3, is a crucial regulatory node in the cellular metabolism. The enzyme is an important modulator regulating the intracellular fructose-2,6-bisphosphate level. PFKFB3 is a bifunctional enzyme with an exceptionally high kinase to phosphatase ratio around 740:1.

Profiling Vaccinium macrocarpon components and metabolites in human urine and the urine ex-vivo effect on Candida albicans adhesion and biofilm-formation.

The aim of this work was to profile, by using an HPLC-MS/MS method, cranberry compounds and metabolites found in human urine after ingestion of a highly standardized cranberry extract (Anthocran®). Two different strategies were adopted for the data analysis: a targeted and an untargeted approach. These strategies allowed the identification of 42 analytes including cranberry components, known metabolites and metabolites hitherto unreported in the literature, including six valerolactones/valeric acid derivatives whose presence in urine after cranberry consumption has never been described before.

Development and validation of a HPLC method for the direct separation of carnosine enantiomers and analogues in dietary supplements.

The chiral purity of some molecules such as nutraceuticals is fundamental to ensure their beneficial activities and it must be checked during quality control analysis. Carnosine is a natural histidine dipeptide used as ingredient for food supplements, but only his L-enantiomer is absorbed and active. Despite of this feature, a method for the separation of carnosine enantiomers without derivatization has only recently been published.

Differentially Expressed Proteins in Primary Endothelial Cells Derived From Patients With Acute Myocardial Infarction.

Endothelial dysfunction is one of the primary factors in the onset and progression of atherothrombosis resulting in acute myocardial infarction (AMI). However, the pathological and cellular mechanisms of endothelial dysfunction in AMI have not been systematically studied. Protein expression profiling in combination with a protein network analysis was used by the mass spectrometry-based label-free quantification approach.

Pro-oxidant and pro-inflammatory effects of glycated albumin on cardiomyocytes.

Human serum albumin (HSA) is the most abundant circulating protein in the body and presents an extensive range of biological functions. As such, it is prone to undergo post-translational modifications (PTMs). The non-enzymatic early glycation of HSA, one of the several PTMs undergone by HSA, arises from the addition of reducing sugars to amine group residues, thus modifying the structure of HSA.

The Disposal of Reactive Carbonyl Species through Carnosine Conjugation: What We Know Now.

Reactive Carbonyl Species are electrophiles generated by the oxidative cleavage of lipids and sugars. Such compounds have been described as important molecules for cellular signaling, whilst their accumulation has been found to be cytotoxic as they may trigger aberrant modifications of proteins (a process often referred to as carbonylation). A correlation between carbonylation of proteins and human disease progression has been shown in ageing, diabetes, obesity, chronic renal failure, neurodegeneration and cardiovascular disease.

Phenolic profiles and anti-inflammatory activities of sixteen table grape (Vitis vinifera L.) varieties.

Several studies have shown that fresh grape and its derivatives contain phenolic compounds exhibiting antioxidant and health promoting effects, particularly in relation to the cardiovascular system. In this study, two methods were developed to characterize sixteen varieties of table and wine grapes: (1) a LC-MS method to identify major and minor phenolic compounds; and (2) a HPLC-DAD method to quantify the most representative compounds. Sixty-seven molecules belonging to different classes of phenolic compounds were identified: anthocyanins, flavan-3-ols, flavonols, stilbenes and organic acids.

Ripe and Raw Pu-Erh Tea: LC-MS Profiling, Antioxidant Capacity and Enzyme Inhibition Activities of Aqueous and Hydro-Alcoholic Extracts.

Herein, we reported a detailed profiling of soluble components of two fermented varieties of Chinese green tea, namely raw and ripe pu-erh. The identification and quantification of the main components was carried out by means of mass spectrometry and UV spectroscopy, after chromatographic separation. The antioxidant capacity towards different radical species, the anti-microbial and the enzyme inhibition activities of the extracts were then correlated to their main constituents.

Development and validation of a sensitive LC-MS/MS assay for the quantification of anserine in human plasma and urine and its application to pharmacokinetic study.

Carnosine (beta-alanyl-L-histidine) and its methylated analogue anserine are present in relevant concentrations in the omnivore human diet. Several studies reported promising therapeutic potential for carnosine in various rodent models of oxidative stress and inflammation-related chronic diseases. Nevertheless, the poor serum stability of carnosine in humans makes the translation of rodent models hard.