Anserine/Carnosine-Rich Extract from Thai Native Chicken Suppresses Melanogenesis via Activation of ERK Signaling Pathway.

Skin hyperpigmentation is an aesthetic problem that leads to psychosocial issues. Thus, skin whitening agents from agro- and poultry-industrial co-products are considered high economic value ingredients of interest for sustainable application. Therefore, this study aimed to determine the cosmeceutical potential of anserine/carnosine-rich chicken extract (ACCE) from the Thai native chicken Pradu Hang Dam Mor Kor 55 (PD) meat. The chemical composition was identified and quantified using the HPLC-UV method. Then, the antioxidation potential of the extract was compared to that of L-anserine and L-carnosine, using 1,1-diphenyl-2-picrylhydrazyl assay and shikonin-induced production of reactive oxygen species in CCD-986Sk cell models, and the anti-melanogenesis effect in the MNT-1 melanoma cell line model was investigated. Furthermore, related mechanisms were identified using colorimetric tyrosinase assay and the Western blot technique. The ACCE was composed of L-anserine and L-carnosine as two major constituents. In a dose-dependent manner, ACCE, L-anserine, and L-carnosine manifested significant antioxidation potential and significant reduction of melanin production. Activation of the extracellular signal-regulated kinase (ERK) signaling pathway and inhibition of tyrosinase activity of ACCE were demonstrated as the mechanisms of the anti-melanogenesis effect. In conclusion, ACCE has been revealed as a potential cosmeceutical agent due to its antioxidation and anti-melanogenic activity in association with L-anserine and L-carnosine composition and biomolecular regulating ability. Therefore, further studies and development should be considered to support the utilization of anserine/carnosine-rich chicken extract in the cosmetic industry for economic value creation and sustainability.

Carnosine and anserine in chicken can quench toxic acrylamide under cooking conditions: Mass spectrometric studies on adduct formation and characterization.

Acrylamide (AA) is a toxic industrial chemical but is also found in heated potato foods such as French fries due to the Maillard reaction between amino acids and reducing sugars. However, high-temperature cooking is often required for flavoring, browning, and sterilizing of raw ingredients. Imidazole dipeptides, such as carnosine (ß-alanyl-l-histidine, CAR) and anserine (ß-alanyl-Nπ-methyl-l-histidine, ANS), are present in high concentrations in meat and are known to scavenge radical species and toxic aldehydes. Here, we investigated the reaction between CAR/ANS and AA under several conditions expected to detoxify AA by cooking with meat. The reaction products were characterized by LC-ESI-MS/MS as CAR/ANS-AA adducts at the N-terminus, and His-Nτ/Nπ. The reactivity of CAR sites toward AA were in the order N-terminus > Nτ > Nπ. A selective LC-ESI-SRM/MS method was also developed and confirmed the formation of CAR/ANS-AA adducts during pan frying of minced potato and chicken breast.

Proximate Composition and Nutritional Profile of Rainbow Trout (Oncorhynchus mykiss) Heads and Skipjack tuna (Katsuwonus Pelamis) Heads.

In order to evaluate the application potential of rainbow trout (Oncorhynchus mykiss) heads and skipjack tuna (Katsuwonus pelamis) heads; proximate composition, amino acids, fatty acids, carnosine, and anserine contents were analyzed in this study. Rainbow trout heads showed significantly higher protein (29.31 g/100 g FW, FW is abbreviation of fresh weight) and lipid (6.03 g/100 g FW) contents than skipjack tuna heads (18.47 g/100 g FW protein and 4.83 g/100 g FW lipid) (p < 0.05). Rainbow trout heads and skipjack tuna heads exhibited similar amino acid composition. Essential amino acids constituted more than 40% of total amino acids in both rainbow trout head and skipjack tuna head. The fatty acid profile was different between rainbow trout heads and skipjack tuna heads. Rainbow trout heads mainly contained 38.64% polyunsaturated fatty acids (PUFAs) and 38.57% monounsaturated fatty acids (MUFAs), whereas skipjack tuna heads mainly contained 54.46% saturated fatty acids (SFAs). Skipjack tuna heads contained 4563 mg/kg FW anserine and 1761 mg/kg FW carnosine, which were both significantly higher than those of rainbow trout heads (p < 0.05). These results demonstrate that both rainbow trout heads and skipjack tuna heads may be used as materials for recycling high-quality protein. Meanwhile, rainbow trout heads can be used to extract oil with high contents of unsaturated fatty acids, while skipjack tuna heads may be a source for obtaining carnosine and anserine.

Simultaneous Detection of Carnosine and Anserine by UHPLC-MS/MS and Its Application on Biomarker Analysis for Differentiation of Meat and Bone Meal.

A novel ultra-high performance liquid chromatography (UHPLC) procedure, coupled with tandem mass spectrometry (MS/MS), was established for the analysis of anserine (ANS) and carnosine (CAR) in meat and bone meal (MBM) (bovine, ovine, porcine, and poultry origins). The pretreatment strategies were optimized for four types of MBM samples prior to UHPLC-MS/MS analysis. This method allowed determining CAR and ANS in short analysis time (18 min per sample). The limits of detection (LODs) and limits of quantification (LOQs) of two analytes in four types of MBM samples were in the ranges of 0.41⁻3.07 ng/g and 0.83⁻5.71 ng/g, respectively. The recovery rates spiked with low, intermediate, and high levels of two analytes in four types of MBM samples were 48.53⁻98.93%, 60.12⁻98.94%, and 67.90⁻98.92%, respectively. Acceptable inter-day reproducibility (RSD < 12.63%) supported the application of this proposed method for determining CAR and ANS in MBM samples. Overall, this rapid, effective, and robust method was successfully applied for quantitative detection of CAR and ANS in MBM samples. Furthermore, The CAR/ANS ratio was found to be in the decreasing order: porcine > bovine > ovine > poultry MBM. This proposed methodology was novelly applied to identify the biomarker (CAR/ANS ratio) for species-specific identification of MBM.

Nutrients Composition in Fit Snacks Made from Ostrich, Beef and Chicken Dried Meat.

The aim of the study was to compare three types of meat snacks made from ostrich, beef, and chicken meat in relation to their nutrients content including fat, fatty acids, heme iron, and peptides, like anserine and carnosine, from which human health may potentially benefit. Dry meat samples were produced, from one type of muscle, obtained from ostrich (m.ambiens), beef (m. semimembranosus), and broiler chicken meat (m. pectoralis major). The composition of dried ostrich, beef, and chicken meat, with and without spices was compared. We show that meat snacks made from ostrich, beef, and chicken meat were characterized by high concentration of nutrients including proteins, minerals (heme iron especially in ostrich, than in beef), biologically active peptides (carnosine-in beef, anserine-in ostrich then in chicken meat). The, beneficial to human health, n-3 fatty acids levels differed significantly between species. Moreover, ostrich jerky contained four times less fat as compared to beef and half of that in chicken. In conclusion we can say that dried ostrich, beef, and chicken meat could be a good source of nutritional components.

Carnosine Catalyzes the Formation of the Oligo/Polymeric Products of Methylglyoxal.

BACKGROUND/AIMS: Reactive dicarbonyl compounds, such as methylglyoxal (MG), contribute to diabetic complications. MG-scavenging capacities of carnosine and anserine, which have been shown to mitigate diabetic nephropathy, were evaluated in vitro and in vivo. METHODS: MG-induced cell toxicity was characterized by MTT and MG-H1-formation, scavenging abilities by Western Blot and NMR spectroscopies, cellular carnosine transport by qPCR and microplate luminescence and carnosine concentration by HPLC. RESULTS: In vitro, carnosine and anserine dose-dependently reduced N-carboxyethyl lysine (CEL) and advanced glycation end products (AGEs) formation. NMR studies revealed the formation of oligo/polymeric products of MG catalyzed by carnosine or anserine. MG toxicity (0.3-1 mM) was dose-dependent for podocytes, tubular and mesangial cells whereas low MG levels (0.2 mM) resulted in increased cell viability in podocytes (143±13%, p<0.001) and tubular cells (129±3%, p<0.001). Incubation with carnosine/anserine did not reduce MG-induced toxicity, independent of incubation times and across large ranges of MG to carnosine/anserine ratios. Cellular carnosine uptake was low (<0.1% in 20 hours) and cellular carnosine concentrations remained unaffected. The putative carnosine transporter PHT1 along with the taurine transporter (TauT) was expressed in all cell types while PEPT1, PEPT2 and PHT2, also belonging to the proton-coupled oligopeptide transporter (POT) family, were only expressed in tubular cells. CONCLUSION: While carnosine and anserine catalyze the formation of MG oligo/polymers, the molar ratios required for protection from MG-induced cellular toxicity are not achievable in renal cells. The effect of carnosine in vivo, to mitigate diabetic nephropathy may therefore be independent upon its ability to scavenge MG and/or carnosine is mainly acting extracellularly.

Computational approaches in the rational design of improved carbonyl quenchers: focus on histidine containing dipeptides.

AIM: The inhibition of protein carbonylation can play therapeutic roles in several oxidative-based diseases and direct carbonyl quenching appears the most effective inhibition strategies. l-carnosine derivatives are effective and selective quenchers toward 4-hydroxy-2-nonenal even though their activity was never investigated in a fully comparable way. RESULTS: The reported results revealed that anserine, homocarnosine and carnosinamide retain a remarkable quenching activity combined with a satisfactory selectivity. In silico analyses confirmed the key role of flexibility, lipophilicity and nucleophilicity parameters in rationalizing the measured reactivity. CONCLUSION: This study confirms that in silico approaches can be successfully used in the rational design of improved carbonyl quenchers. Physicochemical and stereoelectronic descriptors appear really informative especially when explored by their corresponding property spaces.

A vibrational spectroscopy study on anserine and its aqueous solutions.

In this study based on vibrational spectroscopic measurements and Density Functional Theory (DFT), we aimed for a reliable interpretation of the IR and Raman spectra recorded for anserine in the solid phase and water (H2O) and heavy water (D2O) solutions. Initial DFT calculations at the B3LYP/6-31G(d) searched possible conformers of the anserine zwitterion using a systematic conformational search. The corresponding equilibrium geometrical parameters and vibrational spectral data were determined for each of the stable conformers (in water) by the geometry optimization and hessian calculations performed at the same level of theory using the polarized continuum model (PCM). The same calculations were repeated to determine the most energetically preferred dimer structure for the molecule and the associated geometry, force field and vibrational spectral data. The harmonic force constants obtained from these calculations were scaled by the Scaled Quantum Mechanical Force Field (SQM) method and then used in the calculation of the refined wavenumbers, potential energy distributions, IR and Raman intensities. These refined theoretical data, which confirm the zwitterion structure for anserine in the solid phase or aqueous solvents, revealed the remarkable effects of intermolecular hydrogen bonding on the structural properties and observed IR and Raman spectra of this molecule.

Photochemical reaction of 2-(3-benzoylphenyl)propionic acid (ketoprofen) with basic amino acids and dipeptides.

Photoreaction of 2-(3-benzoylphenyl)propionic acid (ketoprofen, KP) with basic amino acids (histidine, lysine, and arginine) and dipeptides (carnosine and anserine) including a histidine moiety in phosphate buffer solution (pH 7.4) has been investigated with transient absorption spectroscopy. With UV irradiation KP(-) gave rise to a carbanion through a decarboxylation reaction, and the carbanion easily abstracted a proton from the surrounding molecule to yield a 3-ethylbenzophenone ketyl biradical (EBPH). The dipeptides as well as the basic amino acids were found to accelerate the proton transfer reaction whereas alanine and glycine had no effect on the reaction, revealing that these amino acids having a protonated side chain act as a proton donor. The formation quantum yield of EBPH was estimated to be fairly large by means of an actinometrical method with benzophenone, and the bimolecular reaction rate constant for the proton transfer between the carbanion and the protonated basic amino acids or the protonated dipeptides was successfully determined. It has become apparent that the bimolecular reaction rate constant for the proton transfer depended on the acid dissociation constant for the side chain of the amino acids for the first time. This reaction mechanism was interpreted by difference of the heat of reaction for each basic amino acid based on the thermodynamical consideration. These results strongly suggest that the side chain of the basic amino acid residue in protein should play an important role for photochemistry of KP in vivo.

Genetic parameters for carnitine, creatine, creatinine, carnosine, and anserine concentration in longissimus muscle and their association with palatability traits in Angus cattle.

The objective of this study was to estimate genetic parameters for carnitine, creatine, creatinine, carnosine, and anserine concentration in LM and to evaluate their associations with Warner-Bratzler shear force (WBSF) and beef palatability traits. Longissimus muscle samples from 2,285 Angus cattle were obtained and fabricated into steaks for analysis of carnitine, creatine, creatinine, carnosine, anserine, and other nutrients, and for trained sensory panel and WBSF assessments. Restricted maximum likelihood procedures were used to obtain estimates of variance and covariance components under a multiple-trait animal model. Estimates of heritability for carnitine, creatine, creatinine, carnosine, and anserine concentrations in LM from Angus cattle were 0.015, 0.434, 0.070, 0.383, and 0.531, respectively. Creatine, carnosine, and anserine were found to be moderately heritable, whereas almost no genetic variation was observed in carnitine and creatinine. Moderate positive genetic (0.25, P < 0.05) and phenotypic correlations (0.25, P < 0.05) were identified between carnosine and anserine. Medium negative genetic correlations were identified between creatine and both carnosine (-0.53, P < 0.05) and anserine (-0.46, P < 0.05). Beef and livery/metallic flavor were not associated with any of the 5 compounds analyzed (P > 0.10), and carnitine concentrations were not associated (P > 0.10) with any of the meat palatability traits analyzed. Carnosine was negatively associated with overall tenderness as assessed by trained sensory panelists. Similar negative associations with overall tenderness were identified for creatinine and anserine. Painty/fishy was the only flavor significantly and negatively associated with creatinine and carnosine. These results provide information regarding the concentration of these compounds, the amount of genetic variation, and evidence for negligible associations with beef palatability traits in LM of beef cattle.

Improved liquid-chromatography tandem mass spectrometry method for the determination of the bioactive dipeptides, carnosine and anserine: application to analysis in chicken broth.

An improved method, based on ultra-performance liquid chromatography (UPLC) coupled to tandem mass spectrometry (MS/MS), has been developed to determine the bioactive dipeptides carnosine (CAR) and anserine (ANS) in chicken broth. These analytes are hydrophilic (polar) and in order to improve their retention, the chromatographic mode used was hydrophilic interaction chromatography (HILIC) (1.7 µm particle size). In order to remove the salt before the chromatographic analysis of the chicken broth (0.8%, w/w), an exhaustive sample pre-treatment strategy was necessary since the salt is not volatilized and could block the ionization source and lead to signal suppression. The chicken broth was firstly centrifuged to remove the fat and chicken proteins, and then was pretreated by off-line solid-phase extraction (SPE), using traditional cartridges, or off-line µElution plate (µSPE), using microplates, and the results were compared. Due to the high polar character of the dipeptides studied and the sample matrix, these compounds were not retained in the sorbent hydrophilic-lipophilic balanced (HLB) and were eluted in the load step, whereas the salt was retained in the sorbent. This fact was observed by the addition of silver nitrate in the chicken broth extract, where before the SPE or µSPE a white precipitate (silver chloride) was formed and after the SPE or µSPE this precipitate was not observed. By using these sample pre-treatment strategies, the extraction recoveries were higher than 80%, and the matrix effect was lower than 12%. Once the improved method was developed, the quality parameters of the method were studied. The LODs and LOQs of the CAR and ANS were lower than 6 and 1.8 µg/l, respectively. Then, the method was applied to analyse a commercial chicken broth. This improved method allowed determining CAR and ANS between 6 and 10mg dipeptide/l chicken broth in 10 min (sample pre-treatment and chromatographic analysis). Therefore, the proposed improved method is concluded to be rapid, sensitive and selective for the determination of polar compounds by MS in samples that contain salt.

Salsolinol, a tetrahydroisoquinoline-derived neurotoxin, induces oxidative modification of neurofilament-L protection by histidyl dipeptides.

Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) is a compound derived from dopamine metabolism and is capable of causing dopaminergic neurodegeneration. Oxidative modification of neurofilament proteins has been implicated in the pathogenesis of neurodegenerative disorders. In this study, oxidative modification of neurofilament-L (NF-L) by salsolinol and the inhibitory effects of histidyl dipeptides on NF-L modification were investigated. When NF-L was incubated with 0.5 mM salsolinol, the aggregation of protein was increased in a time-dependent manner. We also found that the generation of hydroxyl radicals (•OH) was linear with respect to the concentrations of salsolinol as a function of incubation time. NF-L exposure to salsolinol produced losses of glutamate, lysine and proline residues. These results suggest that the aggregation of NF-L by salsolinol may be due to oxidative damage resulting from free radicals. Carnosine, histidyl dipeptide, is involved in many cellular defense processes, including free radical detoxification. Carnosine, and anserine were shown to significantly prevent salsolinol- mediated NF-L aggregation. Both compounds also inhibited the generation of •OH induced by salsolinol. The results indicated that carnosine and related compounds may prevent salsolinol-mediated NF-L modification via free radical scavenging.

Anserine and carnosine determination in meat samples by pure micellar liquid chromatography.

Anserine and carnosine, which are both imidazole dipeptides, are natural antioxidants that are present in some types of meat. A pure micellar liquid chromatographic procedure was developed using a micellar mobile phase of 0.10 M sodium dodecyl sulphate buffered at pH 7, an amino column and UV detection. Three types of stationary phases (C18, phenyl and amino columns) were examined and the procedure was used to determine the two compounds in meat samples. They were completely resolved without any interference from the protein band. Total analysis time was 12 min. The limits of detection (ng/mL) were 71 and 53 for anserine and carnosine, respectively. Calibration curves were constructed on three different days (r>0.998). Repeatability and intermediate precision were evaluated at three different concentrations in meat matrices, the residual standard deviations being below 2.1%. Meat samples of poultry, pork and beef were injected directly into the chromatographic system after extraction in a sodium dodecyl sulphate solution and filtration. The possibility of direct injection using micellar liquid chromatography reduces the cost and time of analyses, and decreases error sources owing to minimised risks of losses and chemical changes in the analytes. Moreover, the selection of a pure mobile phase of sodium dodecyl sulphate allows this procedure to offer a number of advantages, such as non-toxicity, non-flammability, biodegradability and low cost, in comparison with aqueous-organic solvents. Its simplicity, then, makes it a good candidate for application in routine analysis in the area of food control and quality.

Homology modeling of human serum carnosinase, a potential medicinal target, and MD simulations of its allosteric activation by citrate.

Recent biochemical and clinical evidence implicates human serum carnosinase in a variety of pathological conditions, such as neurological disorders and diabetic nephropathy, suggesting that this enzyme is of potential interest as a novel medicinal target. The present study was undertaken with a view to model the serum carnosinase and its catalytic site and to unravel the molecular mechanism by which citrate ions increase the catalytic efficiency of serum carnosinase. A homology model of the enzyme was obtained on the basis of beta-alanine synthetase, and its active center was found to bind known substrates carnosine, homocarnosine, and anserine in a binding mode conducive to catalysis. Citrate ions were shown to bind at only three well-defined sites involving both ion pairs and hydrogen bonds. Molecular dynamics simulations evidenced that citrate binding had a remarkable conformational influence on the 3D structure of carnosinase, increasing the binding affinity (i.e., binding score) of carnosine to the catalytic site. This is one of the first reports documenting the molecular mechanism of an allosteric enzyme activator using MD simulations.

Carnosine and anserine act as effective transglycating agents in decomposition of aldose-derived Schiff bases.

There are numerous publications describing the positive effects of carnosine (beta-alanyl-histidine) and anserine (beta-alanyl-1-N-methyl-histidine) on cell and organ function. Of special interest to us is the fact that these dipeptides act to retard and (in one instance) reverse non-enzymatic glycation. To date, the primary explanation for these anti-glycating effects has been the fact that carnosine and anserine can serve as alternative and competitive glycation targets, thereby protecting proteins from this deleterious process. In this paper, we document another mechanism by which these two peptides can retard or reverse glycation. The process involves decomposition of the very first intermediates of the non-enzymatic glycation cascade (aldosamines a.k.a. Schiff bases) by nucleophilic attack of carnosine and/or anserine on the preformed aldosamine such as glucosyl-lysine. If future research shows this reaction is to be physiologically important, this mechanism could explain some of the beneficial effects of carnosine and anserine as anti-glycating agents.

Intrinsic toxicity of glucose, due to non-enzymatic glycation, is controlled in-vivo by deglycation systems including: FN3K-mediated deglycation of fructosamines and transglycation of aldosamines.

Along with oxygen, glucose is an essential macronutrient for most cells, a source of carbons for biosynthesis and energy. However, alongside this indispensable role for cell survival and growth, glucose is intrinsically toxic by reacting with primary amines such as lysine in proteins in a non-enzymatic glycation process (a.k.a. Maillard reaction) especially important in long-lived, homeothermic organisms where temperatures of 37-44 degrees C accelerate its rate. Products of Maillard reactions are known to have adverse effects on protein function and have been implicated in the development of diabetic complications and possibly in neurodegenerative diseases. Because of the unavoidable nature of non-enzymatic glycation and its deleterious effects, we propose that glucose-utilizing organisms, especially the homeothermic ones, possess mechanisms to control this process at its earliest stages. In the intracellular milieu two such mechanisms are apparent at present; a fructosamine-3-kinase(FN3K)-dependent process which is ubiquitous in all warm-blooded animals and a FN3K-independent deglycation pathway present in all animals, including ones which do not have FN3K, such as insects. We propose that of the two pathways, the FN3K-independent mechanism is more important due to the fact that it breaks down the very first intermediate of the Maillard reaction, the Schiff base (a.k.a aldosamine). We postulate that this, FN3K-independent, deglycation occurs by transglycation, in which carbohydrate moieties of glycated amines, such as glucoselysines on proteins, are removed by intracellular nucleophiles including free amino acids and peptides such as glutathione, carnosine and anserine. Furthermore, we hypothesize that one or more of these nucleophile-aldose adducts, formed as by-products of transglycation, are actively removed from cells by one or more of the multi-drug-resistance [MDR] proteins or similar pumps. In the extracellular space, non-enzymatic glycation and deglycation occur as well. We also postulate that, in that setting, transglycation products are removed from the system by the kidneys or similar excretory organs. Our hypothesis leads to several testable predictions including: The deglycation hypothesis offers new paradigm for thinking about non-enzymatic glycation and diabetic complications and offers possible strategies for intervention in this and possibly other degenerative conditions.

Interaction of soluble peptides and proteins from skeletal muscle with volatile compounds in model systems as affected by curing agents.

The effect of curing agents (salt, glucose, nitrate, nitrite, and ascorbic acid) on the binding of skeletal peptides (carnosine and anserine) and a sarcoplasmic protein (myoglobin) with key flavor compounds (hexanal, octanal, 2-pentanone, 2-methylbutanal, and 3-methylbutanal) has been studied by solid-phase microextraction (SPME). Curing agents had an effect on the interaction process between carnosine and volatile compounds, which was higher than the interactions observed with anserine and myoglobin. Sodium chloride decreased the interaction of volatiles with carnosine except for octanal, which was increased, and 2-pentanone, which was unaltered. Ascorbic acid exerted the highest effect by decreasing the interaction of carnosine with all of the volatile compounds except for octanal and 2-pentanone. The interaction with anserine was affected by sodium chloride, nitrate, and nitrite, producing a decrease in the interaction with hexanal, octanal, and methional. Finally, sodium chloride, glucose, and nitrite increased the interaction of myoglobin with hexanal, octanal, and methional. The effect of simulated stages of the curing process on the binding was also studied. A combined effect of the curing agents resulted in a change in the relative proportions of volatile compounds that can lead to different flavor perceptions of dry-cured meat products.

Carnosine and related dipeptides as quenchers of reactive carbonyl species: from structural studies to therapeutic perspectives.

Carnosine (beta-alanyl-L-histidine) and related peptides such as homocarnosine (gamma-amino-butyryl-histidine), balenine beta-alanyl-L-3-methylhistidine) and anserine beta-alanyl-L-1-methylhistidine) are histidine-containing dipeptides (HD) particularly abundant in excitable tissues such as nervous system and skeletal muscle. Although their biochemical role is still unknown, several evidences indicate that these endogenous compounds act as quenchers of reactive and cytotoxic carbonyl species. In this presentation we will review the structural evidences and ex vivo data supporting this hypothesis. We first elucidated the reaction mechanism of carnosine as quencher of alpha, beta-unsaturated aldehydes such as 4-hydroxy-trans-2,3-nonenal (HNE) and acrolein (ACR) and then demonstrated the efficacy of carnosine and related peptides as detoxifying agents of HNE in spontaneously oxidized rat skeletal muscle, by detecting the corresponding HNE-Michael adducts in the crude biological matrix by liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Finally, we set-up and validated a sensitive, selective and specific LC-ESI-MS/MS method for the determination of HD and of the corresponding HNE-Michael adducts to monitor their profile in physiological (aging) and pathological conditions (diabetes, atherosclerosis) characterized by a carbonyl-mediated degenerative overload. The results obtained, beside to give a contribution to the understanding of the biochemical role of histidine-dipeptides, provide a strong rational to the design of novel derivatives, active as exogenous agents able to detoxify carbonyl compounds.

Gender-related differences in carnosine, anserine and lysine content of murine skeletal muscle.

The aminoacyl-imidazole dipeptides carnosine (beta-alanyl-L-histidine) and anserine (beta-alanyl-1-methyl-histidine) are present in relatively high concentrations in excitable tissues, such as muscle and nervous tissue. In the present study we describe the existence of a marked sexual dimorphism of carnosine and anserine in skeletal muscles of CD1 mice. In adult animals the concentrations of anserine were higher than those of carnosine in all skeletal muscles studied, and the content of aminoacyl-imidazole dipeptides was remarkably higher in males than in females. Postnatal ontogenic studies and hormonal manipulations indicated that carnosine synthesis was up-regulated by testosterone whereas anserine synthesis increased with age. Regional variations in the concentrations of the dipeptides were observed in both sexes, skeletal muscles from hind legs having higher amounts of carnosine and anserine than those present in fore legs or in the pectoral region. The concentration of L-lysine in skeletal muscles also showed regional variations and a sexual dimorphic pattern with females having higher levels than males in all muscles studied. The results suggest that these differences may be related with the anabolic action of androgens on skeletal muscle.

Interactions of soluble peptides and proteins from skeletal muscle on the release of volatile compounds.

The ability of skeletal dipeptides (carnosine and anserine) and a sarcoplasmic protein (myoglobin) to interact with key flavor compounds (hexanal, octanal, methional, 2-pentanone, 2-methylbutanal, and 3-methylbutanal) has been studied using the solid phase microextraction (SPME) technique. Conditions for SPME analysis (fiber coating, sampling time, and linearity of detection) were optimized. The effect of pH on the binding was also investigated. Thermodynamic models were applied to evaluate the binding parameters n (number of binding sites), K (affinity constant), and DeltaG (Gibb's free energy) to all of the flavor compounds studied, and they showed an absence of cooperative effect. Carnosine was the peptide with the highest affinity for all of the volatile compounds except 2-pentanone. Its interaction with hexanal and methional was significantly affected by pH. Anserine showed a lower level of interactions with hexanal, methional, 2-methylbutanal, and 3-methylbutanal, whereas myoglobin interacted with only hexanal and 2-methylbutanal. Differences in aroma retention can thus result in different sensory perceptions of muscle foods.