Genome-wide association studies of anserine and carnosine contents in the breast meat of Korean native chickens.

Histidine-containing dipeptides (HCDs), such as anserine and carnosine, are enormously beneficial to human health and contribute to the meat flavor in chickens. Meat quality traits, including flavor, are polygenic traits with medium to high heritability. Polygenic traits can be improved through a better understanding of their genetic mechanisms. Genome-wide association studies (GWAS) constitute an effective genomic tool to identify the significant single-nucleotide polymorphisms (SNPs) and potential candidate genes related to various traits of interest in chickens. This study identified potential candidate genes influencing the anserine and carnosine contents in chicken meat through GWAS. We performed GWAS of anserine and carnosine using the Illumina chicken 60K SNP chip (Illumina Inc., San Diego, CA) in 637 Korean native chicken-red-brown line (KNC-R) birds consisting of 228 males and 409 females. The contents of anserine and carnosine in breast meat of KNC-R chickens were investigated. The mean value of the anserine and carnosine are 29.12 mM/g and 10.69 mM/g respectively. The genomic heritabilities were moderate (0.24) for anserine and high (0.43) for carnosine contents. Four and nine SNPs were significantly (P < 0.05) associated with anserine and carnosine, respectively. Based on the GWAS result, the 30.6 to 31.9 Mb region on chicken chromosome 7 was commonly associated with both anserine and carnosine. Through the functional annotation analysis, we identified HNMT and HNMT-like genes as potential candidate genes associated with both anserine and carnosine. The results presented here will contribute to the ongoing improvement of meat quality to satisfy current consumer demands, which are based on healthier, better-flavored, and higher-quality chicken meat.

Anserine is expressed in human cardiac and skeletal muscles.

We evaluated whether anserine, a methylated analog of the dipeptide carnosine, is present in the cardiac and skeletal muscles of humans and whether the CARNMT1 gene, which encodes the anserine synthesizing enzyme carnosine-N-methyltransferase, is expressed in human skeletal muscle. We found that anserine is present at low concentrations (low micromolar range) in both cardiac and skeletal muscles, and that anserine content in skeletal muscle is ~15 times higher than in cardiac muscle (cardiac muscle: 10.1 ± 13.4 µmol·kg-1 of dry muscle, n = 12; skeletal muscle: 158.1 ± 68.5 µmol·kg-1 of dry muscle, n = 11, p < 0.0001). Anserine content in the heart was highly variable between individuals, ranging from 1.4 to 45.4 µmol·kg-1 of dry muscle, but anserine content was not associated with sex, age, or body mass. We also showed that CARNMT1 gene is poorly expressed in skeletal muscle (n = 10). This is the first study to demonstrate that anserine is present in the ventricle of the human heart. The presence of anserine in human heart and the confirmation of its expression in human skeletal muscle open new avenues of investigation on the specific and differential physiological functions of histidine dipeptides in striated muscles.

l-Anserine Increases Muscle Differentiation and Muscle Contractility in Human Skeletal Muscle Cells.

l-Anserine, an imidazole peptide, has a variety of physiological activities, but its effects on skeletal muscle differentiation and muscle contractile force remain unknown. Thus, in this study, we investigated the effect of l-anserine on muscle differentiation and muscle contractile force in human skeletal muscle cells. In two-dimensional culture, 1 µM l-anserine significantly increased the myotube diameters (26.5 ± 1.71, 27.7 ± 1.08, and 28.8 ± 0.85 µm with 0, 0.1, and 1 µM l-anserine, respectively) and the expression levels of genes involved in muscle differentiation and the sarcomere structure. In three-dimensional culture, 1 µM l-anserine significantly increased the contractile force of engineered human skeletal muscle tissues cultured on a microdevice (1.99 ± 0.30, 2.17 ± 0.62, 2.66 ± 0.39, and 3.28 ± 0.85 µN with 0, 0.1, 0.5, and 1 µM l-anserine, respectively). l-Anserine also increased the myotube diameters and the proportion of myotubes with sarcomere structures in the cultured tissues. Furthermore, the histamine receptor 1 (H1R) antagonist attenuated the l-anserine-induced increase in the contractile force, suggesting the involvement of H1R in the mechanism of action of l-anserine. This study showed for the first time that l-anserine enhances muscle differentiation and muscle contractility via H1R.

Desorption electrospray ionization-mass spectrometry imaging of carnitine and imidazole dipeptides in pork chop tissues.

Carnitine is essential for energy production and lipid metabolism in skeletal muscle. Carnosine and its methylated analogs anserine and balenine are histidine-containing imidazole dipeptides, which are antioxidative compounds. They are major health-related components in meat; however, analytical technique to investigate their distribution among tissues have not fully established. Here, we performed desorption electrospray ionization (DESI)-mass spectrometry imaging (MSI) of pork chop sections containing longissimus thoracis et lumborum muscle (loin), intermuscular fat tissue, transparent tissue, and spinalis muscle to investigate the distributions of carnitine and imidazole dipeptides. Liquid chromatography-MS revealed that the concentrations of carnitine, carnosine, anserine, and balenine were 11.0 ± 0.9, 330.1 ± 15.5, 21.2 ± 1.5, and 9.6 ± 0.5 mg/100 g, respectively. In the mass spectrum obtained by DESI-MSI, peaks corresponding to the chemical formulae of carnitine and imidazole dipeptides were detected. DESI-MSI provided definite identification of carnitine, while DESI-tandem MSI (MS/MSI) was necessary to accurately visualize carnosine, anserine, and balenine. Carnitine and these imidazole dipeptides were mainly distributed in the loin and spinalis muscle, while their distribution was not uniform in both muscle tissues. In addition, the balance between both tissues were different. The concentration of carnitine was higher in the spinalis muscle than that in the loin, while those of imidazole dipeptides were higher in the loin than those in the spinalis muscle. These results were consistent with those obtained by liquid chromatography-MS quantification, suggest that DESI-MSI analysis is useful for the distribution analysis of carnitine and imidazole dipeptides in meat.

A t-test ranking-based discriminant analysis for classification of free-range and barn-raised broiler chickens by 1H NMR spectroscopy.

Intensive systems of raising chickens in barns prevail worldwide for financial reasons. In contrast, free-range chickens are raised in better welfare conditions, and preferred by consumers due to their distinctive taste/flavor, having higher market prices. Thus, free-range chickens have been the target of frauds. In this study, 1H NMR metabolic profiles of breasts of free-range and barn-raised broilers (108 individuals) were compared by two discriminant models, based on t-test ranking and partial least squares (PLS-DA). Both models provided 100 % of correct classification in both training and test sets, being the univariate model based on t-test screening simpler and more robust. Among other differences, barn-raised broilers presented lower carnosine and anserine concentrations, and higher free amino acids contents. Univariate discrimination was based on the ratio of two NMR signals assigned to ß-alanine and carnosine + anserine, respectively. As an additional advantage, this profiling method could be adapted to other measurement platforms.

In vivo detection of carnosine and its derivatives using chemical exchange saturation transfer.

PURPOSE: To detect carnosine, anserine and homocarnosine in vivo with chemical exchange saturation transfer (CEST) at 17.2 T. METHODS: CEST MR acquisitions were performed using a CEST-linescan sequence developed in-house and optimized for carnosine detection. In vivo CEST data were collected from three different regions of interest (the lower leg muscle, the olfactory bulb and the neocortex) of eight rats. RESULTS: The CEST effect for carnosine, anserine and homocarnosine was characterized in phantoms, demonstrating the possibility to separate individual contributions by employing high spectral resolution (0.005 ppm) and low CEST saturation power (0.15 µ$$ \mu $$ T). The CEST signature of these peptides was evidenced, in vivo, in the rat brain and skeletal muscle. The presence of carnosine and anserine in the muscle was corroborated by in vivo localized spectroscopy (MRS). However, the sensitivity of MRS was insufficient for carnosine and homocarnosine detection in the brain. The absolute amounts of carnosine and derivatives in the investigated tissues were determined by liquid chromatography-electrospray ionization-tandem mass spectrometry using isotopic dilution standard methods and were in agreement with the CEST results. CONCLUSION: The robustness of the CEST-linescan approach and the favorable conditions for CEST at ultra-high magnetic field allowed the in vivo CEST MR detection of carnosine and related peptides. This approach could be useful to investigate noninvasively the (patho)-physiological roles of these molecules.

Effects of ß-alanine and L-histidine supplementation on carnosine contents in and quality and secondary structure of proteins in slow-growing Korat chicken meat.

Carnosine enrichment of slow-growing Korat chicken (KRC) meat helps differentiate KRC from mainstream chicken. We aimed to investigate the effects of ß-alanine and L-histidine supplementation on the carnosine synthesis in and quality and secondary structure of proteins in slow-growing KRC meat. Four hundred 21-day-old female KRC were used, and a completely randomized design was applied. The chickens were divided into 4 experimental groups: basal diet (A), basal diet supplemented with 1.0% ß-alanine (B), 0.5% L-histidine (C), and 1.0% ß-alanine combined with 0.5% L-histidine (D). Each group consisted of 5 replicates (20 chickens per replicate). On d 70, 2 chickens per replicate were slaughtered, and the levels of carnosine, anserine, and thiobarbituric acid reactive substances were analyzed. Biochemical changes were monitored using synchrotron radiation-based Fourier transform infrared microspectroscopy; 5 chickens per replicate were slaughtered, and the meat quality was analyzed. Statistical analysis was performed using ANOVA and principal component analysis (PCA). Group D chickens exhibited the highest carnosine meat content, followed by those in groups B and C. However, amino acid supplementation did not affect anserine content and growth performance. Higher carnosine levels correlated with increasing pH45 min and decreasing drip loss, cooking loss, shear force, and lipid oxidation. PCA revealed that supplementation with only ß-alanine or L-histidine was related to increased content of ß-sheets, ß-turns, and aliphatic bending groups and decreased content of α-helix groups. This study is the first to report such findings in slow-growing chicken. Our findings suggest that KRC can synthesize the highest carnosine levels after both ß-alanine and L-histidine supplementation. Higher carnosine contents do not adversely affect meat quality, improve meat texture, and alter the secondary structures of proteins. The molecular mechanism underlying carnosine synthesis in chickens needs further study to better understand and reveal markers that facilitate the development of nutrient selection programs.

Oral anserine supplementation does not attenuate type-2 diabetes or diabetic nephropathy in BTBR ob/ob mice.

Carnosine, a naturally occurring dipeptide present in an omnivorous diet, has been shown to ameliorate the development of metabolic syndrome, type-2 diabetes (T2D) and early- and advanced-stage diabetic nephropathy in different rodent models. Anserine, its methylated analogue, is more bio-available in humans upon supplementation without affecting its functionality. In this work, we investigated the effect of oral supplementation with anserine or carnosine on circulating and tissue anserine and carnosine levels and on the development of T2D and diabetic nephropathy in BTBR ob/ob mice. BTBR ob/ob mice were either supplemented with carnosine or anserine in drinking water (4 mM) for 18 weeks and compared with non-supplemented BTBR ob/ob and wild-type (WT) mice. Circulating and kidney, but not muscle, carnosine, and anserine levels were enhanced by supplementation with the respective dipeptides in ob/ob mice compared to non-treated ob/ob mice. The evolution of fasting blood glucose, insulin, fructosamine, triglycerides, and cholesterol was not affected by the supplementation regimens. The albumin/creatine ratio, glomerular hypertrophy, and mesangial matrix expansion were aggravated in ob/ob vs. WT mice, but not alleviated by supplementation. To conclude, long-term supplementation with anserine elevates circulating and kidney anserine levels in diabetic mice. However, anserine supplementation was not able to attenuate the development of T2D or diabetic nephropathy in BTBR ob/ob mice. Further research will have to elucidate whether anserine can attenuate milder forms of T2D or metabolic syndrome.

Proteolysis and protein oxidation throughout the smoked dry-cured ham process.

During the five stages of smoked dry-cured ham processing, proteolysis and protein oxidation were simultaneously detected in the Biceps femoris (BF) and Semimembranosus (SM) muscles. Proteolysis was more advanced in BF than in SM throughout the process of production. The total FAA increased significantly (p < 0.05) throughout the processing, resulting in higher total FAA content in BF than in SM muscle. SDS-PAGE revealed progressive degradation of sarcoplasmic proteins of investigated muscles, with the pronounced changes for the 69.9-41.7 kDa region. SDS-PAGE of BF showed more intense degradation of myofibrillar proteins due to greater proteolysis in BF. Electrophoresis of myofibrillar proteins evidenced the marked degradation of 130 kDa, 96.7 kDa and 27-20.7 kDa bands in both muscles. A similar trend was observed for protein oxidation in BF and SM, with the final values of 26.36 and 23.7 nmol carbonyls/mg proteins, respectively. The Pearson correlation revealed a strong relationship between protein oxidation and proteolysis.

Potential of 2D qNMR spectroscopy for distinguishing chicken breeds based on the metabolic differences.

Two-dimensional quantitative NMR spectroscopy (2D qNMR) was set up and multivariate analyses were performed on metabolites obtained from breast meat extracts of broilers and four native chicken (KNC) strains. It can accurately identify more metabolites than 1D 1H NMR via separation of peak overlap by dimensional expansion with good linearity, but has a problem of numerical quantification; Complementation of 1D and 2D qNMR is necessary. Among breeds, KNC-D had higher amounts of free amino acids, sugars, and bioactive compounds than others. Noticeable differences between KNCs and broilers were observed; KNCs contained higher amounts of inosine 5'-monophosphate, α-glucose, anserine, and lactic acid, and lower amounts of free amino acids and their derivatives. The 2D qNMR combined with multivariate analyses distinguished the breast meat of KNCs from broilers but showed similarities among KNCs. Also, 2D qNMR may provide fast metabolomics information compared to conventional analysis.

The anserine to carnosine ratio: an excellent discriminator between white and red meats consumed by free-living overweight participants of the PREVIEW study.

BACKGROUND: Biomarkers of meat intake hold promise in clarifying the health effects of meat consumption, yet the differentiation between red and white meat remains a challenge. We measure meat intake objectively in a free-living population by applying a newly developed, three-step strategy for biomarker-based assessment of dietary intakes aimed to indicate if (1) any meat was consumed, (2) what type it was and (3) the quantity consumed. METHODS: Twenty-four hour urine samples collected in a four-way crossover RCT and in a cross-sectional analysis of a longitudinal lifestyle intervention (the PREVIEW Study) were analyzed by untargeted LC-MS metabolomics. In the RCT, healthy volunteers consumed three test meals (beef, pork and chicken) and a control; in PREVIEW, overweight participants followed a diet with high or moderate protein levels. PLS-DA modeling of all possible combinations between six previously reported, partially validated, meat biomarkers was used to classify meat intake using samples from the RCT to predict consumption in PREVIEW. RESULTS: Anserine best separated omnivores from vegetarians (AUROC 0.94-0.97), while the anserine to carnosine ratio best distinguished the consumption of red from white meat (AUROC 0.94). Carnosine showed a trend for dose-response between non-consumers, low consumers and high consumers for all meat categories, while in combination with other biomarkers the difference was significant. CONCLUSION: It is possible to evaluate red meat intake by using combinations of existing biomarkers of white and general meat intake. Our results are novel and can be applied to assess qualitatively recent meat intake in nutritional studies. Further work to improve quantitation by biomarkers is needed.

Ameliorative effects of functional chalaza hydrolysates prepared from protease-A digestion on cognitive dysfunction and brain oxidative damages.

Our patented protease A-digested crude chalaza hydrolysates (CCH) show antioxidant abilities in vitro. The prophylactic effects of CCH on cognitive dysfunction and brain oxidative damages were investigated via a D-galactose (DG)-injected mouse model in this study. Fifty-four mice were randomly divided into the following: (1) CON, 0.1 mL 0.9% saline (subcutaneous injection [SC] on the back)+distilled water (oral gavage); (2) DG, 100 mg/kg BW/day D-galactose (Bio-Serv Co., Flemington, NJ, USA) (SC on the back)+distilled water (oral gavage); (3) DG_LCH, 100 mg/kg BW/day D-galactose (SC on the back) + 50 mg CCH/kg BW/day in 0.1 ml distilled water (oral gavage); (4) DG_MCH, 100 mg/kg BW/day D-galactose (SC on the back) + 100 mg CCH/kg BW/day (oral gavage); (5) DG_HCH, 100 mg/kg BW/day D-galactose (SC on the back) + 200 mg CCH/kg BW/day (oral gavage); (6) DG_AG, 100 mg/kg BW/day D-galactose (SC on the back) + 100 mg aminoguanidine hydrochloride/kg BW/day (oral gavage). The experiment lasted for 84 D. CCH, containing antioxidant-free amino acids and anserine, restored (P < 0.05) DG-injected memory injury in the Morris water maze test and attenuated the neuronal degenerations and nucleus shrinkages in the dentate gyrus area. CCH supplementation also reduced amyloid ß-peptide protein levels and accumulation of advanced glycation end products (AGE) in the brain of DG-injected mice, whereas the brain antioxidant capacity was reversed (P < 0.05) by supplementing CCH. Furthermore, AGE receptor (RAGE), NFκb, IL-6, and TNF-α gene expressions were downregulated (P < 0.05) by supplementing CCH. Therefore, CCH show prophylactic effects on the development of oxidative stress-induced cognitive dysfunction.

Comparison of the quality characteristics of chicken breast meat from conventional and animal welfare farms under refrigerated storage.

In this study, we aimed to investigate the meat quality characteristics, bioactive compound content, and antioxidant activity during refrigerated storage of breast meat of Arbor Acres broilers (carcass weight: 1.1 kg, raised for 35 D) obtained from a conventional farm (BCF, n = 30) and an animal welfare farm (BAF, n = 30) in Korea. The BCF and BAF did not differ in their proximate composition, color, water-holding capacity, creatine, creatinine, and carnosine contents. However, the shear force value was significantly higher in BAF than in BCF (P < 0.05). The 2-thiobarbituric acid reactive substance (TBARS) levels in BCF on days 7 and 9 were significantly higher than those in BAF (P < 0.001). During storage, the total volatile basic nitrogen (VBN) content of BAF was significantly lower, except on day 1. The fatty acid composition of samples was not affected by the storage period, however, saturated fatty acid and unsaturated fatty acid contents did differ among the types of farm systems (P < 0.05). Although the creatine, creatinine, and carnosine contents in BAF and BCF did not differ significantly, the carnosine and creatinine contents decreased with the increase in storage period (P < 0.05). The anserine content of BAF was significantly higher than that of BCF throughout storage. Superoxide dismutase activity was not affected by the type of farm system but was affected by storage period. Overall, BAF showed lower pH, microorganism, TBARS, and VBN values, and higher anserine contents than BCF. These findings can serve as reference data for the evaluation of chicken meat quality of broilers raised in animal welfare farm and conventional farm.

Composition of amino acids and related nitrogenous nutrients in feedstuffs for animal diets.

We analyzed the composition of amino acids (AAs) in oligopeptides, proteins, and the free pool, as well as creatine, agmatine, polyamines, carnosine, anserine, and glutathione, in animal- and plant-derived feedstuffs. Ingredients of animal origins were black soldier fly larvae meal (BSFM), chicken by-product meal, chicken visceral digest, feather meal, Menhaden fishmeal, Peruvian anchovy fishmeal, Southeast Asian fishmeal, spray-dried peptone from enzymes-treated porcine mucosal tissues, poultry by-product meal (pet-food grade), spray-dried poultry plasma, and spray-dried egg product. Ingredients of plant origins were algae spirulina meal, soybean meal, and soy protein concentrate. All animal-derived feedstuffs contained large amounts of all proteinogenic AAs (particularly glycine, proline, glutamate, leucine, lysine, and arginine) and key nonproteinogenic AAs (taurine and 4-hydroxyproline), as well as significant amounts of agmatine, polyamines, creatine, creatinine, creatine phosphate, and glutathione. These nitrogenous substances are essential to either DNA and protein syntheses in cells or energy metabolism in tissues (particularly the brain and skeletal muscle). Of note, chicken by-product meal, poultry by-product meal, and spray-dried poultry plasma contained large amounts of carnosine and anserine (potent antioxidants). Compared with most of the animal-derived feedstuffs, plant-derived feedstuffs contained much lower contents of glycine and proline, little 4-hydroxyproline, and no creatine, creatinine, creatine phosphate, carnosine or anserine. These results indicate the unique importance of animal-source feedstuffs in improving the feed efficiency, growth and health of animals (including fish and companion animals). Because soy protein concentrate is consumed by infants, children and adults, as are BSFM and algae for children and adults, our findings also have important implications for human nutrition.

Metabolite profile based on 1H NMR of broiler chicken breasts affected by wooden breast myodegeneration.

The objective was to characterize the effect of wooden breast (WB) myodegeneration on the metabolite profile of chicken meat by 1H NMR and multivariate data analysis. The results displayed that the metabonome of chicken breast consisted predominantly of 30 metabolites, including amino acids, organic acids, carbohydrates, alkaloids, nucleosides and their derivatives. WB-affected samples showed higher leucine, valine, alanine, glutamate, lysine, lactate, succinate, taurine, glucose, and 5'-IMP levels, but lower histidine, ß-alanine, acetate, creatine, creatinine, anserine and nicotinamide adenine dinucleotide levels compared to normal fillets (p < 0.05). In conclusion, results indicated that WB-affected fillets possessed a unique biochemical signature. This unique profile could identify candidate biomarkers for diagnostic utilization and provide mechanistic insight into biochemical processes leading to WB myopathy in commercial broiler chickens.

Differences in muscle histidine-containing dipeptides in broilers.

BACKGROUND: Poultry meat has high levels of histidine-containing dipeptides (HCD) and consumption of meat rich in HCD may elicit certain health benefits. The aim of this work was to compare the HCD content (anserine and carnosine) in the breast and thigh muscles of two broiler strains differing in growth rate, feeding regime, and age at slaughter. A 3 (production system) × 2 (sex) × 2 (age at slaughter) full factorial arrangement was applied with fast-growing Ross 308 chicks fed ad libitum (ROSS-AL), slow-growing Sasso T451 chicks fed ad libitum (SASSO-AL), and Ross 308 chicks given limited feeding (ROSS-LIM). At the age of 40 and 62 days, eight birds per production system × sex combination were randomly selected for sampling of the breast and thigh muscle. Muscle HCD content was determined by high-performance liquid chromatography (HPLC). RESULTS: Across treatments, levels of anserine were 2.5- and 1.9-fold higher than carnosine in breast and thigh muscle respectively (P < 0.001), and levels of anserine and carnosine were 2.2- and 2.8-fold higher respectively in breast versus thigh muscle (P < 0.001). In breast muscle, SASSO-AL had higher levels of HCD than ROSS-AL and ROSS-LIM (P < 0.001). Considering different market meat types, breast muscle of 62-day-old SASSO-AL birds had more than threefold higher content of HCD compared to thigh muscle of 40-day-old ROSS-AL birds (P < 0.001). CONCLUSION: Large differences in muscle HCD content were found, varying according to type of muscle and broiler. © 2019 Society of Chemical Industry.

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. Even though anserine and carnosine have similar biochemical properties, anserine has better serum stability. Despite this interesting profile, the research on anserine is scarce. The aim of this study was to explore the bioavailability and stability of synthesized anserine by (1) performing in vitro stability experiments in human plasma and molecular modelling studies and by (2) evaluating the plasma and urinary pharmacokinetic profile in healthy volunteers following different doses of anserine (4-10-20 mg/kg body weight). A bio-analytical method for measuring anserine levels was developed and validated using liquid chromatography-electrospray mass spectrometry. Both plasma (CMAX: 0.54-1.10-3.12 µM) and urinary (CMAX: 0.09-0.41-0.72 mg/mg creatinine) anserine increased dose-dependently following ingestion of 4-10-20 anserine mg/kg BW, respectively. The inter-individual variation in plasma anserine was mainly explained by the activity (R2 = 0.75) and content (R2 = 0.77) of the enzyme serum carnosinase-1. Compared to carnosine, a lower interaction energy of anserine with carnosinase-1 was suggested by molecular modelling studies. Conversely, the two dipeptides seems to have similar interaction with the PEPT1 transporter. It can be concluded that nutritionally relevant doses of synthesized anserine are well-absorbed and that its degradation by serum carnosinase-1 is less pronounced compared to carnosine. This makes anserine a good candidate as a more stable carnosine-analogue to attenuate chronic diseases in humans.

[Determination of anserine, homocarnosine and carnosine in meat products by ion chromatography with integrated pulsed amperometric detection].

A method was developed for the simultaneous determination of anserine, homocarnosine and carnosine in meat samples using ion chromatography (IC) with integrated pulsed amperometric detection (IPAD). The samples were separated on a high performance anion exchange AminoPac PA10 column (250 mm×2 mm) using 100 mmol/L NaOH as the eluent. The flow rate was kept at 0.2 mL/min and the column temperature was set at 30℃. The three target compounds were separated within 15 min, and there were no interferences from 17 tested amino acids in their determination. Under the optimal chromatographic conditions, anserine, homocarnosine and carnosine showed good linearity in the range of 0.05-5 mg/L with correlation coefficients (r) greater than 0.99. The limits of detection (LODs) and limits of quantification (LOQs) were in the range of 8.9-22.1 µg/L and 29.6-73.6 µg/L, respectively. The proposed method was applied to the analysis of duck breast and goose breast meat samples. The average spiked recoveries ranged from 92.4% to 104.5%. This simple and sensitive method can be applied to the determination of related nutrients in meat products.

A simple and reliable new microchip electrophoresis method for fast measurements of imidazole dipeptides in meat from different animal species.

Microchip electrophoresis (ME) was applied for the separation of two physiologically important imidazole dipeptides-carnosine and anserine. The capacitively coupled contactless conductivity detector (C4D) was employed for quantification of both dipeptides after separation in a new home-built ME unit. The separation parameters were optimized as follows to enable quantitative, baseline separation of both dipeptides: injection time 16 s, injection voltage 900 V/cm, and separation voltage 377.1 V/cm. The C4D detector responded linearly to both imidazole dipeptides in the range 0-20 mg L-1. The known addition methodology was applied to test the accuracy of the measurement of imidazole dipeptides in a complex sample. The recoveries for measurement of carnosine in the mixture ranged from 96.1 to 105.0%, whereas those for anserine amounted to 96.6 to 102.0%. This method was also applied to real biological samples. The results exhibited a satisfactory agreement with a standard HPLC method. The proposed ME method represents a cheap, fast, and simple alternative to the existing, more complicated and expensive HPLC methods. This method does not demand either the optical detectors nor tedious derivatization of sample, which are unavoidable in HPLC methods. The method was succesfuly applied for animal species determination in unknown meat samples using the carnosine/anserine ratio, and subsequently, it could be used in a food fraud prevention process. Graphical abstract Microchip electrophoresis portable device with a C4D detector for determination of imidazole dipeptides in model samples and real meat samples from different animal species.

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.