Imidazole Dipeptides Research Articles

Determination of Imidazole Dipeptides and Related Amino Acids in Natural Seafoods by Liquid Chromatography-Tandem Mass Spectrometry Using a Pre-Column Derivatization Reagent.

Imidazole dipeptides (IDPs) and taurine (Tau) have several health benefits and are known to be contained in natural seafoods. However, their levels vary widely in different natural seafoods, making their simultaneous determination desirable. Herein, we employ a liquid chromatography-tandem mass spectrometry approach using a novel amino group derivatization reagent, succinimidyl 2-(3-((benzyloxy)carbonyl)-1-methyl-5-oxoimidazolidin-4-yl) acetate ((R)-CIMa-OSu), for the simultaneous quantification of IDPs (carnosine (Car) and anserine (Ans)), their related amino acids, and Tau in natural seafoods. Each seafood sample contained different concentrations of IDPs (Car: ND to 1.48 mmol/100 g-wet, Ans: ND to 4.67 mmol/100 g-wet). The Car levels were considerably higher in eel, while Tau was more abundant in squid, boiled octopus, and scallop. Thus, the derivatization reagent (R)-CIMa-OSu provides a new approach to accurately assess the nutritional composition of seafoods, thereby providing valuable insight into its dietary benefits.

Anserine, a Histidine-Containing Dipeptide, Suppresses Pressure Overload-Induced Systolic Dysfunction by Inhibiting Histone Acetyltransferase Activity of p300 in Mice.

Anserine, an imidazole dipeptide, is present in the muscles of birds and fish and has various bioactivities, such as anti-inflammatory and anti-fatigue effects. However, the effect of anserine on the development of heart failure remains unknown. We cultured primary cardiomyocytes with 0.03 mM to 10 mM anserine and stimulated them with phenylephrine for 48 h. Anserine significantly suppressed the phenylephrine-induced increases in cardiomyocyte hypertrophy, ANF and BNP mRNA levels, and histone H3K9 acetylation. An in vitro histone acetyltransferase (HAT) assay showed that anserine directly suppressed p300-HAT activity with an IC50 of 1.87 mM. Subsequently, 8-week-old male C57BL/6J mice were subjected to transverse aortic constriction (TAC) and were randomly assigned to receive daily oral treatment with anserine-containing material, Marine Active® (60 or 200 mg/kg anserine) or vehicle for 8 weeks. Echocardiography revealed that anserine 200 mg/kg significantly prevented the TAC-induced increase in left ventricular posterior wall thickness and the decrease in left ventricular fractional shortening. Moreover, anserine significantly suppressed the TAC-induced acetylation of histone H3K9. These results indicate that anserine suppresses TAC-induced systolic dysfunction, at least in part, by inhibiting p300-HAT activity. Anserine may be used as a pharmacological agent for human heart failure therapy.

Functional analysis of the carnosine synthase-like gene conserved in Monoraphidium braunii

Carnosine, a dipeptide composed of β-alanine and histidine, is predominantly found in animals such as mammals, birds, and fish, with no known presence in plants, microalgae, and other organisms. It can undergo methylation to produce anserine, β-alanyl-N-methyl-L-histidine. These imidazole peptides are known for their antioxidative properties and fatigue recovery effects. The synthesis of carnosine is catalyzed by carnosine synthase, encoded by the ATPGD1 or CARNS1 gene. Recently, homologous proteins to carnosine synthase have been reported from several microalgal genomes, although their biological functions remain unknown. In this study, we investigated the role of carnosine synthase-like protein in the green alga Monoraphidium braunii SAG 48.87. Initially, we examined the accumulation of carnosine and anserine in M. braunii cells cultivated under various conditions. Interestingly, salt stress induced the accumulation of anserine. The cells treated with 420 mM NaCl accumulated 38.3 μg g−1 dry weight (DW) of anserine. Expression of ATPGD1a, putatively annotated as a carnosine synthase gene, was induced under salt stress conditions, while expression of ATPGD1b was downregulated. The Escherichia coli cells expressing the recombinant ATPGD1a primarily accumulated anserine (269.9 μg g−1 DW) and a small amount of carnosine, although we could not confirm any carnosine or anserine synthesis by the purified recombinant protein. These findings suggest that the carnosine synthase homolog in microalgae may play a role in synthesizing imidazole dipeptides, and anserine could serve as a salt protective agent and help stabilize cellular functions, particularly under salt conditions. This is the first observation of the synthesis of the imidazole dipeptide from any organism except animals and anserine protecting from salt stress.

Comparison of physiologically functional compounds in Sika deer Cervus nippon meats obtained from different regions in Japan.

In Japan, the promotion of effective use of many wild deer as food resource has been conducted. However, they are not necessarily utilized effectively. Thus, we focused physiologically functional compounds to find characteristics of Sika deer meats (commercially available) obtained from different regions such as Hokkaido, Wakayama, Tokushima, and Miyazaki prefectures in Japan, making it a valuable resource for future studies and applications. The amount of carnosine, anserine, and balenine in muscle of deer from Wakayama prefecture was significantly lower than that in muscle of deer from other prefectures. The differences of amount of imidazole dipeptides in different prefectures seems to be caused by feed, rearing environment, and breed. The amount of carnitine in deer meat from Hokkaido was significantly lower than that in muscle of deer from other prefectures, while the amount of acetyl-carnitine in deer meat from Miyazaki prefectures was significantly higher than that from other prefectures. The amounts of glutamine, ornithine, and 3-methylhistidine in muscles of deer from Wakayama prefectures were significantly higher than those in muscle of deer from other prefectures. These results might be caused by differences in feeding habits, habitat, the muscle types, and subspecies of deer obtained from four regions in Japan.

Effects of Cyclic High Ambient Temperature on Muscle Imidazole Dipeptide Content in Broiler Chickens.

Imidazole dipeptides possess important bioregulatory properties in animals. This study aimed to evaluate the effect of high ambient temperature on muscle imidazole dipeptides (carnosine, anserine, and balenine) in broiler chickens. Sixteen 14-day-old male broiler chickens were divided into two groups, which were reared under thermoneutral (25 ± 1 °C) or cyclic high ambient temperature (35 ± 1 °C for 8 h/day) for 4 weeks. Chickens exposed to cyclic high ambient temperatures displayed lower skeletal muscle anserine and carnosine content than control chickens. Balenine could not be detected in the pectoral muscle of either group. The pectoral muscles of broiler chickens kept under cyclic high-temperature exhibited significantly lower mRNA expression of carnosine synthase 1, which synthesizes carnosine and anserine; but a significantly higher mRNA expression of carnosinase 2, which degrades carnosine and anserine. Our results suggest that heat exposure decreases pectoral imidazole dipeptide content in broiler chickens. This may be attributed to a lower expression of imidazole dipeptide-synthesizing genes, but higher levels of genes involved in their degradation.

Effects of sturgeon fillet intake on top-ranked Japanese female long-distance runners.

The aim of this study is to investigate whether consumption of sturgeon fillets reduces the oxidative stress marker urinary 8-hydroxy-2'-deoxyguanosine (8OHdG) in top-ranked Japanese female long-distance runners. In a before-and-after study, nine professional long-distance female athletes ate 100 g/day of sturgeon fillets for 2 weeks. Urinalysis (8OHdG, an oxidative stress marker, and creatinine), blood tests (fatty acids and 25-hydroxyvitamin D [25OHD]), exercise intensity, subjective fatigue, muscle elasticity, muscle mass, body fat mass, and nutritional intake using image-based dietary assessment (IBDA) were compared before, immediately after, and 1 month after the intervention. Consumption of sturgeon fillets suppressed 8OHdG (p < 0.05) in the increased exercise intensity female athletes. Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and 25OHD levels in blood increased from before to immediately after and 1 month after the intervention (p < 0.05). IBDA showed that intake of n-3 fatty acid increased after and one month after the intervention, whereas DHA, imidazole dipeptide and vitamin D intake increased after the intervention (p < 0.05) and then decreased after 1 month (p < 0.05). There were no significant changes in subjective fatigue, muscle elasticity, muscle mass, and body fat. The results suggest that eating sturgeon fillets during intense training may increase blood levels of EPA, DHA, and 25OHD, which may suppress urinary oxidative stress (8OHdG) in top-ranked Japanese long-distance runners.

Composition and taste of beef, pork, and duck meat and bioregulatory functions of imidazole dipeptides in meat

This study quantified the nutritional components and imidazole dipeptide levels of commercially available meats (beef, pork, and duck), and their effects on taste were quantified via taste recognition devices. Although meat and its products are considered high-risk diets, meat components, such as imidazole dipeptides, exert bioregulatory functions. Further, considering their bioregulatory function, commercial meats’ antioxidant activity and vascular endothelial function were examined. Characteristic variations in nutritional components were observed depending on the type and part of meat analyzed. These components affected the taste and texture of meat. The main imidazole dipeptides detected were anserine (duck meat) and carnosine (beef and pork). Meat with larger quantities of total imidazole dipeptide demonstrated better sensory test results. Therefore, anserine and carnosine effects on taste were determined using a taste recognition device; carnosine alone produced a noticeably bitter taste, whereas adding anserine reduced bitterness and enhanced umami taste. In a few cases, cooking enhanced the quantity of carnosine and/or anserine and their antioxidant activities. We demonstrated the ability of imidazole dipeptides, particularly anserine, to improve nitric oxide production in vascular endothelial cells. This study provides essential information for health-conscious consumers to develop high-quality, functional meat products.

Improving the enzymatic activity of l-amino acid α-ligase for imidazole dipeptide production by site-directed mutagenesis.

Imidazole dipeptides, histidine-containing dipeptides, including carnosine (β-alanyl-l-histidine), anserine (β-alanyl-3-methyl-l-histidine), and balenine (β-alanyl-1-methyl-l-histidine) in animal muscles have physiological functions, such as significant antioxidant and antifatigue effects. They are obtained by extraction from natural raw materials, including chicken and fish meat. However, using natural raw materials entails stable supply and mass production limitations. l-amino acid α-ligase (Lal) catalyzes the formation of various dipeptides from unprotected l-amino acids by conjugating with adenosine 5'-triphosphate (ATP) hydrolysis reaction. In this study, site-directed mutagenesis of Lal was applied to establish an efficient method for producing imidazole dipeptides by the enzymatic process. We significantly improved the conversion rate from substrate amino acids compared with wild-type Lal.

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.

The short-term feeding of low- and high-histidine diets prior to market affects the muscle carnosine and anserine contents and meat quality of broilers.

Functional dipeptides carnosine and anserine are abundant in muscle. We determined the effect of short-term dietary histidine (His) content on muscle carnosine and anserine contents and meat quality of broilers. Three groups of 28-day-old female broilers were fed diets with His contents of 67%, 100%, or 150% of requirement for 10days before market (His contents 0.21%, 0.32%, and 0.48%, respectively). The carnosine and anserine contents of 0-h aged muscle significantly increased with dietary His content; in particular, the carnosine content was 162% higher in the His 0.48% group than in the His 0.32% group. The contents of both peptides also increased with dietary His content in 48-h aged muscle, but carnosine was not detected in 0- and 48-h aged muscle of the His 0.21% group. The drip loss, cooking loss, shear force, and pH of meat were not affected by the dietary His content. The 2-thiobarbituric acid-reactive substances contents of 24- and 48-h aged muscles were lower in the His 0.48% group than in the other groups, and the a* and b* values were lower in the His 0.21% group. These results suggest that short-term dietary His content affects imidazole dipeptide contents, antioxidative capacity, and color of broiler meat.

Effects of Inherent Lactic Acid Bacteria on Inhibition of Angiotensin I-Converting Enzyme and Antioxidant Activities in Dry-Cured Meat Products.

The aim of this study was to investigate the inherent bacteria that contribute to expressing the angiotensin I-converting enzyme (ACE) inhibitory activity and the antioxidant activity of dry-cured meat products without a bacterial starter. Among the ten dry-cured meat product samples, Coppa and Milano salami exhibited high ACE inhibitory activity, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability, and oxygen radical absorbance capacity (ORAC). No consistent trend was observed in the pH values or the total peptide and imidazole dipeptide concentration of the products that exhibited high ACE inhibitory and antioxidant activities in the tested samples. To investigate the bacteria contributing to the ACE inhibitory and antioxidant activities of the product, 16S rRNA sequencing analysis, isolation, and identification of bacteria were performed using not only Coppa and Milano salami but also the Jamon Serrano and Parma prosciutto products that had low functional activities. Results suggest the Lactobacillales order, particularly the species Latilactobacillus sakei and Pediococcus pentosaceus, were the main inherent bacteria in Coppa and Milano salami, respectively, compared with the Jamon Serrano and Parma prosciutto products. Therefore, the inherent lactic acid bacteria in dry-cured meat products without bacterial starter is important for ACE inhibitory and antioxidant activities of the products.

Pharmacokinetics and tissue distribution of orally administrated imidazole dipeptides in carnosine synthase gene knockout mice.

Imidazole dipeptides (ID) are abundant in skeletal muscle and the brain and have various functions, such as antioxidant, pH-buffering, metal-ion chelation. However, the physiological significance of ID has not been fully elucidated. In this study, we orally administered ID to conventional carnosine synthase gene-deficient mice (Carns-KO mice) to investigate the pharmacokinetics. Carnosine or anserine was administered at a dose of 500 mg (∼2 mmol) per kilogram of mouse body weight, and ID contents in the tissues were measured. No ID were detected in untreated Carns-KO mice. In the ID treatment groups, the ID concentrations in the tissues increased in a time-dependent manner in the gastrocnemius muscle, soleus muscle, and cerebrum after ID administration. Our findings suggest that the Carns-KO mice are a valuable animal model for directly evaluating the effects of dietary ID and for elucidating the physiological functions of oral ID administration.

Balenine, Imidazole Dipeptide Promotes Skeletal Muscle Regeneration by Regulating Phagocytosis Properties of Immune Cells.

Balenine is one of the endogenous imidazole dipeptides derived from marine products. It is composed of beta-alanine and 3-methyl-L-histidine, which exist mainly in the muscles of marine organisms. The physiological functions of dietary balenine are not well-known. In this study, we investigated whether the supplementation of dietary balenine was associated with muscle function in a cardiotoxin-indued muscle degeneration/regeneration model. Through morphological observation, we found that the supplementation of balenine-enriched extract promoted the regeneration stage. In addition, the expression of regeneration-related myogenic marker genes, such as paired box protein 7, MyoD1, myogenin, and Myh3, in a group of mice fed a balenine-enriched extract diet was higher than that in a group fed a normal diet. Moreover, the supplementation of balenine-enriched extract promoted the expression of anti-inflammatory cytokines as well as pro-inflammatory cytokines at the degeneration stage. Interestingly, phagocytic activity in the balenine group was significantly higher than that in the control group in vitro. These results suggest that balenine may promote the progress of muscle regeneration by increasing the phagocytic activity of macrophages.

Metabolism of Imidazole Dipeptides, Taurine, Branched-Chain Amino Acids, and Polyamines of the Breast Muscle Are Affected by Post-Hatch Development in Chickens.

To explore metabolic characteristics during the post-hatch developmental period, metabolomic analyses of breast muscle and plasma were performed in chickens. The most significant growth-related changes in metabolite levels were observed between seven and 28 days of age. Some of these metabolites are essential nutrients or reported as growth-promoting metabolites. In the muscle, two imidazole dipeptides—carnosine and its methylated metabolite, anserine—increased with the development. These dipeptide levels may be, in part, regulated transcriptionally because in the muscle mRNA levels of carnosine synthase and carnosine methylation enzyme increased. In contrast, taurine levels in the muscle decreased. This would be substrate availability-dependent because some upstream metabolites decreased in the muscle or plasma. In branched-chain amino acid metabolism, valine, leucine, and isoleucine decreased in the muscle, while some of their downstream metabolites decreased in the plasma. The polyamines, putrescine and spermidine, decreased in the muscle. Furthermore, mRNA levels associated with insulin/insulin-like growth factor 1 signaling, which play important roles in muscle growth, increased in the muscle. These results indicate that some metabolic pathways would be important to clarify metabolic characteristics and/or growth of breast muscle during the post-hatch developmental period in chickens.

Development of a quantitative method for analyzing three imidazole dipeptides using high-performance liquid chromatography and its application for meat and fish

Imidazole dipeptides (IDPs) such as anserine, balenine, and carnosine are functional peptides that are expected to improve cognitive functions and have antioxidant properties. However, there are few methods to extract these three representative IDPs from foods with high-efficiency and analyze them simultaneously using simple, rapid, and accurate high-performance liquid chromatography (HPLC). In this study, an analysis method was developed based on the combination of an internal standard, an extraction reagent, and HPLC conditions for the quantification of IDPs. IDPs in foods were extracted using 8 % perchloric acid solution with 4-imidazoleacrylic acid added as an internal standard; the IDPs were then analyzed by isocratic elution using an amino column. IDPs in nine types of meat and fish were quantified. The recovery and the quantification ranges of IDPs on the established method were 97.4 %–103.3 % and 0.78 μM–200 μM, respectively. The results showed that the amounts of balenine, anserine, and carnosine were extremely high in whale, salmon, and pork, respectively. Interestingly, venison contained the three IDPs, and the ratio of anserine to carnosine (balenine:anserine:carnosine = 1:4:5) was similar, which differed in other animal meats. This method can be used effectively, particularly for the quantification of foods containing these three IDPs.

Metabolomic and pharmacologic analyses of brain substances associated with sleep pressure in mice

Sleep pressure, the driving force of the homeostatic sleep regulation, is accumulated during wakefulness and dissipated during sleep. Sleep deprivation (SD) has been used as a method to acutely increase animal’s sleep pressure for investigating the molecular changes under high sleep pressure. However, SD induces changes not only reflecting increased sleep pressure but also inevitable stresses and prolonged wake state itself. The Sik3Sleepy mutant mice (Sleepy) exhibit constitutively high sleep pressure despite sleeping longer, and have been useful as a model of increased sleep pressure. Here we conducted a cross-comparison of brain metabolomic profiles between SD versus ad lib slept mice, as well as Sleepy mutant versus littermate wild-type mice. Targeted metabolome analyses of whole brains quantified 203 metabolites in total, of which 43 metabolites showed significant changes in SD, whereas three did in Sleepy mutant mice. The large difference in the number of differential metabolites highlighted limitations of SD as methodology. The cross-comparison revealed that a decrease in betaine and an increase in imidazole dipeptides are associated with high sleep pressure in both models. These metabolites may be novel markers of sleep pressure at the whole-brain level. Furthermore, we found that intracerebroventricular injection of imidazole dipeptides increased subsequent NREM sleep time, suggesting the possibility that imidazole dipeptides may participate in the regulation of sleep in mice.

Emerging cardioprotective mechanisms of vitamin B6: a narrative review.

Although overt vitamin B6 deficiency is rare, marginal vitamin B6 deficiency is frequentandoccurs in a consistent proportion of the population. The marginal vitamin B6 deficiency appears to relate to an increased risk of inflammation-related diseases, such as cardiovascular diseases and cancers.Ofallthecardiovascular diseases,heart failure is a complex clinical syndrome associated with a high mortality rate. So far, information regarding the cardioprotective mechanisms of vitamin B6 has been limited. Meanwhile, recent studies have revealed that vitamin B6 treatment increases cardiac levels of imidazole dipeptides (e.g.,carnosine, anserine, and homocarnosine), histamine, and γ-aminobutyric acid (GABA) and suppresses P2X7 receptor-mediated NLRP3 inflammasome. These modulations may imply potentialcardioprotective mechanisms ofvitamin B6. These modulationsmayalsobe involved in the underlying mechanismsthroughwhich vitamin B6 suppresses oxidative stress and inflammation. This review provides an up-to-date evaluation of our current understanding of the cardioprotective mechanisms of vitamin B6.

Isolation of balenine from opah (Lampris megalopsis) muscle and comparison of antioxidant and iron-chelating activities with other major imidazole dipeptides

Balenine (Bal) in opah muscle was extracted using hot water and purified by ion-exchange chromatography and recrystallization to provide 41 g of over 95% pure Bal from 1 kg of opah muscle. The structure of purified Bal was identical to that of an authentic Bal standard by NMR analysis. The antioxidant (ORAC and HORAC values) and Fe(II) ion-chelating abilities of purified Bal were examined by comparison with two major imidazole dipeptides, carnosine (Car) and anserine (Ans). Opah-derived Bal showed significantly higher ORAC and HORAC values and Fe(II) ion-chelating ability at 0.3 mM. In silico molecular simulation revealed that Bal and Car formed hydrogen bonds between the hydrogen atom of the imidazole imino group and the carboxyl carbonyl oxygen, whereas Ans did not. The proposed method for extracting and purifying Bal from opah muscle suggests that opah can be utilized as a functional food or Bal resource.

Balenine, imidazole dipeptide, induces activation of superoxide dismutase in myotubes

Balenine is one of the endogenous imidazole dipeptides. It is composed of beta-alanine and 3-methyl-l-histidine, which exist mainly in the muscles and the brain. The exact biological properties of balenine are still not well known, although the antioxidant activity of carnosine, another imidazole dipeptide, is known. In this study we investigated whether balenine exhibits antioxidant activity. It was found to decrease the superoxide anion (O2−) and increased hydrogen peroxide (H2O2) generation. We found that SOD activity increased in balenine-treated C2C12 myotubes, although balenine did not increase expression of SOD mRNA. On the other hand, there were no changes in other antioxidant enzymes, CAT and GPX activity, and mRNA levels. In an in vitro assay, the direct activation of SOD treated by balenine was significantly higher than with carnosine treatment. Moreover, balenine constituent amino acids did not have the ability to activate SOD. Our results suggest that balenine contributes to antioxidant effects through activation of SOD.

Influence of Imidazole-Dipeptides on Cognitive Status and Preservation in Elders: A Narrative Review.

The worldwide increase in the number of patients with dementia is becoming a growing problem, while Alzheimer’s disease (AD), a primary neurodegenerative disorder, accounts for more than 70% of all dementia cases. Research on the prevention or reduction of AD occurrence through food ingredients has been widely conducted. In particular, histidine-containing dipeptides, also known as imidazole dipeptides derived from meat, have received much attention. Imidazole dipeptides are abundant in meats such as poultry, fish, and pork. As evidenced by data from recent human intervention trials conducted worldwide, daily supplementation of carnosine and anserine, which are both imidazole dipeptides, can improve memory loss in the elderly and reduce the risk of developing AD. This article also summarizes the latest researches on the biochemical properties of imidazole dipeptides and their effects on animal models associated with age-related cognitive decline. In this review, we focus on the results of human intervention studies using supplements of poultry-derived imidazole dipeptides, including anserine and carnosine, affecting the preservation of cognitive function in the elderly, and discuss how imidazole dipeptides act in the brain to prevent age-related cognitive decline and the onset of dementia.