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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. Human genetic diseases with altered carnosinase activity have been identified and associated with neurological disorders and age-related cognitive decline. On the contrary, low peripheral carnosinase activity has been associated with kidney protection, especially in diabetic nephropathy. Therefore, serum carnosinase is a druggable target for the development of selective inhibitors. However, only one molecule (i.e., carnostatine) has been discovered with the purpose of developing serum carnosinase inhibitors. Bestatin is the only inhibitor reported other than carnostatine, although its activity is not selective towards serum carnosinase. Herein, we present a review of the most critical findings on human serum carnosinase, including enzyme expression, localization and substrate selectivity, along with factors affecting the hydrolytic activity, its implication in human diseases and the properties of known inhibitors of the enzyme. [ABSTRACT FROM AUTHOR]

Sugar- and lipid-derived aldehydes are reactive carbonyl species (RCS) frequently used as surrogate markers of oxidative stress in obesity. A pathogenic role for RCS in metabolic diseases of obesity remains controversial, however, partly because of their highly diffuse and broad reactivity and the lack of specific RCS-scavenging therapies. Naturally occurring histidine dipeptides (e.g., anserine and carnosine) show RCS reactivity, but their therapeutic potential in humans is limited by serum carnosinases. Here, we present the rational design, characterization, and pharmacological evaluation of carnosinol, i.e., (2S)-2-(3-amino propanoylamino)-3-(1H-imidazol-5-yl)propanol, a derivative of carnosine with high oral bioavailability that is resistant to carnosinases. Carnosinol displayed a suitable ADMET (absorption, distribution, metabolism, excretion, and toxicity) profile and was determined to have the greatest potency and selectivity toward [alpha],[beta]-unsaturated aldehydes (e.g., 4-hydroxynonenal, HNE, ACR) among all others reported thus far. In rodent models of diet-induced obesity and metabolic syndrome, carnosinol dose-dependently attenuated HNE adduct formation in liver and skeletal muscle, while simultaneously mitigating inflammation, dyslipidemia, insulin resistance, and steatohepatitis. These improvements in metabolic parameters with carnosinol were not due to changes in energy expenditure, physical activity, adiposity, or body weight. Collectively, our findings illustrate a pathogenic role for RCS in obesity-related metabolic disorders and provide validation for a promising new class of carbonyl-scavenging therapeutic compounds rationally derived from carnosine., IntroductionOvernutrition from fatty acids and complex carbohydrates is known to cause oxidative stress from multiple enzymatic and nonenzymatic sources due to the high caloric content and the prevalence of these [...]

Carnosinase (i.e., CN1; E.C. 3.4.13.20) is an enzyme found in the sera of higher primates. CN1 preferentially catalyzes the hydrolysis of natural, orally adsorbed histidine dipeptides like carnosine (i.e., β-alanyl-L-histidine). This is the reason why carnosine has a limited use as a human food supplement or pharmacological agent, despite the promising results obtained in experiments on animal models of human diseases. Herein, an assay is reported for the measurement of serum CN1 activity. The method is intended for the screening of CN1 inhibitors able to enhance carnosine bioavailability in humans. The method was developed to monitor serum hydrolytic activity via the quantitation of one of the products of carnosine hydrolysis (i.e., histidine). Separation was achieved without using organic solvents by means of ion chromatography (IC), whereas detection was provided by UV spectroscopy. The assay herein reported is a green and cost-effective alternative to a recently published method based on hydrophilic interaction liquid chromatography (HILIC) and mass spectrometry (MS). The results show that such a method produces reliable measurements of serum hydrolytic activity and can be used for the screening of CN1 inhibitors. [ABSTRACT FROM AUTHOR]

Human exposure to dicarbonyls occurs via ingestion (e.g., food), inhalation (e.g., electronic cigarettes) and dysregulation of endogenous metabolic pathways (e.g., glycolysis). Dicarbonyls are electrophiles able to induce carbonylation of endogenous substrate. They have been associated with the onset and progression of several human diseases. Several studies have advocated the use of dicarbonyl binders as food preservatives or as drugs aimed at mitigating carbonylation. This study presents the setup of an easy and cheap assay for the screening of selective and potent dicarbonyl binders. The method is based on the incubation of the candidate molecules with a molecular probe. The activity is then determined by measuring the residual concentration of the molecular probe over time by liquid chromatography (LC). However, the naturally occurring dicarbonyls (e.g., glyoxal, methylglyoxal) are not appealing as probes since they are hard to separate and detect using the most popular LC variants. Benzylglyoxal (BGO) was therefore synthesized and tested, proving to be a convenient probe that allows a direct quantification of residual dicarbonyls by reversed phase LC without derivatization. The method was qualified by assessing the binding ability of some molecules known as binders of natural occurring dicarbonyls, obtaining results consistent with literature. [ABSTRACT FROM AUTHOR]

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. Sampling and sample pretreatment miniaturisation were achieved thanks to the development of a volumetric dried blood microsampling approach. Volumetric absorptive microsampling (VAMS) allows the accurate sampling of microvolumes of blood with advantages including, but not limited to, minimal sampling invasiveness, logistical improvements, method sustainability in terms of solvents and energy consumption, and improvement of animal studies in the framework of the 3Rs (Replacement, Reduction and Refinement) principles on animal welfare. The VAMS-LC-MS/MS method exhibited good selectivity, and correlation coefficient values for the calibration curves of each analyte were >0.9987. The limits of quantitation ranged from 0.1 to 25 ng/mL. The intra- and inter-day precisions in terms of RSD were <9.6%. All analytes were stable in whole blood VAMS samples stored at room temperature for at least 30 days with analyte losses < 14%. The developed method was successfully applied to the analysis of biological samples from mice, leading to the unambiguous determination of all the considered target analytes. This method can therefore be applied to analyse TRP metabolites and related biomarkers levels to monitor disease states, perform mechanistic studies and investigate the outcomes of therapeutic interventions. [ABSTRACT FROM AUTHOR]

Cyclo(His-Pro) (CHP) is a cyclic dipeptide which is endowed with favorable pharmacokinetic properties combined with a variety of biological activities. CHP is found in a number of protein-rich foods and dietary supplements. While being stable at physiological pH, CHP can open yielding two symmetric dipeptides (His-Pro, Pro-His), the formation of which might be particularly relevant from dietary CHP due to the gastric acidic environment. The antioxidant and protective CHP properties were repeatedly reported although the non-enzymatic mechanisms were scantly investigated. The CHP detoxifying activity towards α,β unsaturated carbonyls was never investigated in detail, although its open dipeptides might be effective as already observed for histidine containing dipeptides. Hence, this study investigated the scavenging properties of TRH, CHP and its open derivatives towards 4-hydroxy-2-nonenal. The obtained results revealed that Pro-His possesses a marked activity and is more reactive than l-carnosine. As investigated by DFT calculations, the enhanced reactivity can be ascribed to the greater electrophilicity of the involved iminium intermediate. These findings emphasize that the primary amine (as seen in l-carnosine) can be replaced by secondary amines with beneficial effects on the quenching mechanisms. Serum stability of the tested peptides was also evaluated, showing that Pro-His is characterized by a greater stability than l-carnosine. Docking simulations suggested that its hydrolysis can be catalyzed by serum carnosinase. Altogether, the reported results evidence that the antioxidant CHP properties can be also due to the detoxifying activity of its open dipeptides, which might be thus responsible for the beneficial effects induced by CHP containing food. [ABSTRACT FROM AUTHOR]

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. Moreover, carnosine has also been found to act indirectly by activating the Nrf2 transcription factor, a mechanism that would explain many of the effects evoked by this peptide such as anti-inflammatory, antioxidant, antiglycation and anti-carbonyl effects and taken together would explain its therapeutic effect. The present review reports and discusses the most recent studies on the molecular mechanisms of carnosine which need to be fully clarified before promoting carnosine and derivatives as therapeutic agents. [ABSTRACT FROM AUTHOR]

Featured Application: Exploration of Lactobacillus helveticus strains as source of bioactive peptides for potential applications as functional foods as well as for potential uses in nutraceuticals. Among various food sources, milk proteins remain the major vector for functional peptides endowed with several biological activities. Particularly, the proteolytic activity of lactic acid bacteria during milk fermentation has been one of the most followed strategies to produce bioactive peptides. In the present study, the exploration of the activity of several starter cultures, at different fermentation times, was firstly investigated by reversed phase-high performance liquid chromatography. Among the tested strains, Lactobacillus helveticus showed a higher proteolytic activity and it was submitted to further investigations by changing the fermentation substrate (skim milk, brain heart infusion, peptone water) as well as the extraction strategy (trichloroacetic acid vs. glass beads). The chromatographic analyses and the in vitro antioxidant and antihypertensive assays highlighted considerable differences for L. helveticus hydrolysates from different substrates, while a negligible impact by the two extraction protocols emerged. Furthermore, nano-high pressure liquid chromatography coupled with a high resolution mass spectrometry analyzer allowed the preliminary discrimination of fractions from fermented skim milk, likely responsible for the found activity. The obtained results suggest the possibility of varying the fermentation parameters in order to maximize the functional effects of the bioactive peptides. [ABSTRACT FROM AUTHOR]

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. These changes may affect HSA functions impairing its biological activity, finally leading to cell damage. The aim of this study was to quantitate glycated-HSA (GA) levels in the plasma of heart failure (HF) patients and to evaluate the biological effects of GA on HL-1 cardiomyocytes. Plasma GA content from HF patients and healthy subjects was measured by direct infusion electrospray ionization mass spectrometry (ESI-MS). Results pointed out a significant increase of GA in HF patients with respect to the control group (p < 0.05). Additionally, after stimulation with GA, proteomic analysis of HL-1 secreted proteins showed the modulation of several proteins involved, among other processes, in the response to stress. Further, stimulated cells showed a rapid increase in ROS generation, higher mRNA levels of the inflammatory cytokine interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α), and higher levels of the oxidative 4-HNE-protein adducts and carbonylated proteins. Our findings show that plasma GA is increased in HF patients. Further, GA exerts pro-inflammatory and pro-oxidant effects on cardiomyocytes, which suggest a causal role in the etiopathogenesis of HF. Image 1 • Early glycation of HSA may occur under normo- and hyperglycemic conditions. • GA levels increase in HF and positively correlates with disease severity. • GA exerts pro-inflammatory and pro-oxidant effects on cardiomyocytes. [ABSTRACT FROM AUTHOR]

Reactive carbonyl species (RCS) are cytotoxic molecules that originate from lipid peroxidation and sugar oxidation. Natural derivatives can be an attractive source of potential RCS scavenger. However, the lack of analytical methods to screen and identify bioactive compounds contained in complex matrices has hindered their identification. The sequestering actions of various rice extracts on RCS have been determined using ubiquitin and 4-hydroxy-2-nonenal (HNE) as a protein and RCS model, respectively. Black rice with giant embryo extract was found to be the most effective among various rice varieties. The identification of bioactive compounds was then carried out by an isotopic signature profile method using the characteristic isotopic ion cluster generated by the mixture of HNE: 2H5-HNE mixed at a 1:1 stoichiometric ratio. An in-house database was used to obtain the structures of the possible bioactive components. The identified compounds were further confirmed as HNE sequestering agents through HPLC-UV analysis. [ABSTRACT FROM AUTHOR]

Reactive intermediate deaminase (Rid) protein family is a recently discovered group of enzymes that is conserved in all domains of life and is proposed to play a role in the detoxification of reactive enamines/imines. UK114, the mammalian member of RidA subfamily, was identified in the early 90s as a component of perchloric acid-soluble extracts from goat liver and exhibited immunomodulatory properties. Multiple activities were attributed to this protein, but its function is still unclear. This work addressed the question of whether UK114 is a Rid enzyme. Biochemical analyses demonstrated that UK114 hydrolyzes α-imino acids generated by L- or D-amino acid oxidases with a preference for those deriving from Ala > Leu = L-Met > L-Gln, whereas it was poorly active on L-Phe and L-His. Circular Dichroism (CD) analyses of UK114 conformational stability highlighted its remarkable resistance to thermal unfolding, even at high urea concentrations. The half-life of heat inactivation at 95 °C, measured from CD and activity data, was about 3.5 h. The unusual conformational stability of UK114 could be relevant in the frame of a future evaluation of its immunogenic properties. In conclusion, mammalian UK114 proteins are RidA enzymes that may play an important role in metabolism homeostasis also in these organisms. [ABSTRACT FROM AUTHOR]

Abstract: Carnosine is present in high concentrations in specific human tissues such as the skeletal muscle, and among its biological functions, the remarkable scavenging activity toward reactive carbonyl species is noteworthy. Although the two enantiomers show almost identical scavenging reactivity toward reactive carbonyl species, only d‐carnosine is poorly adsorbed at the gastrointestinal level and is stable in human plasma. Direct methods for the enantioselective analysis of carnosine are still missing even though they could find more effective applications in the analysis of complex matrices. In the present study, the use of two different chiral stationary phases is presented. A chiral ligand‐exchange chromatography stationary phase based on N,S‐dioctyl ‐d‐penicillamine resulted in the direct enantioseparation of carnosine. Indeed, running the analysis at 25°C and 1.0 mL/min with a 1.5 mM copper(II) sulfate concentration allowed us to obtain separation and resolution factors of 3.37 and 12.34, respectively. However, the use of a copper(II)‐containing eluent renders it hardly compatible with mass spectrometry detectors. With the teicoplanin‐based stationary phase, a mass spectrometry compatible method was successfully developed. Indeed, a water/methanol 60:40 v/v pH 3.1 eluent flowed at 1.0 mL/min and with a 25°C column temperature produced separation and resolution factors of 2.60 and 4.16, respectively. [ABSTRACT FROM AUTHOR]