Determination of Antihypertensive Small Peptides, Val-Tyr and Ile-Val-Tyr, by Fluorometric High-Performance Liquid Chromatography Combined with a Double Heart-Cut Column-Switching Technique

Ueno, Takao; Tanaka, Mitsuru; Matsui, Toshiro; Matsumoto, Kiyoshi

doi:10.2116/analsci.21.997

Cited by 12 publications

(9 citation statements)

References 15 publications

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“…The membrane barrier and transport ability of cells were maintained during the 60 min-transport experiments without any reduction of TEER value (>100 cm 2 ) by Gly-Sar. The amount of Gly-Sar transported to the basolateral side was determined by a fluorescent NDA-derivatization method [14]. Briefly, 10 μL of 20 mM sodium cyanide in borate buffer (pH 9.5) was added to the basolateral solution taken at each time point (100 μL) in a 96-well microplate, followed by the addition of 50 μL of 0.1 mM NDA solution in methanol.…”

Section: Methodsmentioning

confidence: 99%

Benzotropolone moiety in theaflavins is responsible for inhibiting peptide-transport and activating AMP-activated protein kinase in Caco-2 cells

Park

Kunitake

Matsui

2013

FFHD

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Objective: In the small intestine, peptide transporter 1 (PEPT1) plays a role in the transport of di- and tri-peptides. Recently, we found that theaflavins (TFs), dimeric catechins, inhibited the transport of di-peptides across Caco-2 monolayers by suppressing the expression of PEPT1 through AMP-activated protein kinase (AMPK) activation. In this study, we investigated the structural requirement of theaflavins for the effect, and the mechanism(s) underling theaflavin-induced AMPK activation.Methods: Theaflavin-3’-O-gallate (TF3’G) was used for this study, since it possessed the most potent inhibition power for peptide-transport among theaflavins. Absorption ability was measured with Caco-2 cell monolayers treated with or without 20 μM sample (TF3’G or its related compounds) in an Ussing Chamber. The amount of Gly-Sar (a model of PEPT1-transporing peptide) transport at fixed time-points to 60 min was determined by fluorescent naphthalene-2,3-dicarboxaldehyde-derivatized assay (Ex/Em: 405 nm/460 nm). The apparent permeability coefficient (Papp) was used to evaluate the permeability. Expression of PEPT1 protein in Caco-2 cells treated with or without 20 μM TF3’G in the presence or absence of inhibitor (10 μM compound C as AMPK inhibitor or 25 μM STO-609 as CaMKK inhibitor) was evaluated by Western blot.Results: The Papp value of Gly-Sar significantly (P < 0.05) decreased in 20 μM purprogallin-treated Caco-2 cells as well as in TF3’G-treated cells, together with the reduction of PEPT1 expression, while their monomeric catechins did not show any Papp reduction. In TF3'G-treated Caco-2 cells, the recovery of the reduced PEPT1 expression was found by 10 μM compound C, but not STO-609.Conclusion: The study demonstrated that the benzotropolone moiety in theaflavins was a crucial structural requirement for exerting the inhibition of intestinal peptide-transport, and the suppression of PEPT1 expression by theaflavins would be caused by activating LKB1/AMPK pathway, but not CaMKK/AMPK pathway.Keywords: Theaflavin-3’-Ο-gallate, Peptide transport, PEPT1, Benzotropolone, AMP-activated protein kinase, Calmodulin-dependent protein kinase kinase

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Section: Methodsmentioning

confidence: 99%

Benzotropolone moiety in theaflavins is responsible for inhibiting peptide-transport and activating AMP-activated protein kinase in Caco-2 cells

Park

Kunitake

Matsui

2013

FFHD

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“…Since three 13 C-di-peptides responsible for anti-hypertensive action [2,7] possess a structural similarity with Tyr moiety, the selection of packaging materials to separate each di-peptide must be greatly important. In our reports on isolation of ACE inhibitory peptides from natural proteins, C 18 reversed-phase materials in combination to a CH 3 CN-water mobile phase system was found to be the most retentive for small peptides [10,11]. In this study, hence we primarily examined whether a good separation of three 13…”

Section: Optimization Of Lc-mrm-ms/ms Analysismentioning

confidence: 98%

“…into blood system, we have established a fluorimetric heart-cut column-switching high performance liquid chromatography (HPLC) method [10,11], with which an intact absorption of anti-hypertensive peptide, Val-Tyr, into human blood system at a picomole level was demonstrated [12,13]. However, the proposed specific assay for bioactive peptides would provide great effort, because it still requires tedious pre-extraction and pre-separation steps prior to the assay.…”

mentioning

confidence: 99%

Application of 13C stable isotope labeling liquid chromatography–multiple reaction monitoring–tandem mass spectrometry method for determining intact absorption of bioactive dipeptides in rats

Nakashima

Kudo

Iwaihara

et al. 2011

Analytical Biochemistry

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The liquid chromatography-multiple reaction monitoring-tandem mass spectrometry (LC-MRM-MS/MS) method using (13)C stable isotope-labeled dipeptides was newly developed to simultaneously determine the absorption of three antihypertensive peptides (Val-Tyr, Met-Tyr, and Leu-Tyr) into blood of spontaneously hypertensive rats in one run-in assay. After extracting (13)C-labeled peptides in blood sample with a C(18) cartridge, the extract was applied to a (13)C monoisotopic transition LC-MRM-MS/MS system with D-Val-Tyr included as internal standard. An excellent separation of each dipeptide in LC was achieved at the elution condition of 5-100% methanol in 0.1% formic acid at a flow rate of 0.25 ml/min. The (13)C-labeled peptides ionized by electron spray were detected in the positive ion mode within 15 min. The established method showed high reproducibility with less than 10% coefficient of variation as well as high accuracy of more than 85%. After the administration of a mixture containing the three (13)C-labeled dipeptides to rats at each dose of 30 mg/kg, we could successfully determine the intact absorption of each (13)C-labeled peptide with the maximal absorption amount of 1.1 ng/ml plasma for Val-Tyr by the proposed LC-MRM-MS/MS method.

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“…Finally, the sequence identification of the peptides presents in two of these fractions was performed by MALDI-TOF. This analysis allowed identifying novel peptide sequences 4–8 amino acids long, such as NIDMLRL, LVRW, VRWS, VR, and CIHNIVY, containing amino acids and short sequences whose ACE inhibitory capacity had been demonstrated in other systems ( Ueno et al., 2005 ; De Gobba et al., 2014 ; Aluko, 2015b ; Balgir and Sharma, 2017 ) ( Table 1 ).…”

Section: Bioactive Peptides As Blood Pressure Regulatorsmentioning

confidence: 99%

Amaranth as a Source of Antihypertensive Peptides

et al. 2020

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Amaranth is an ancestral crop used by pre-Columbian cultures for 6000 to 8000 years. Its grains have a relevant chemical composition not only from a nutritional point of view but also due to the contribution of components with good techno-functional properties and important potential as bioactive compounds. Numerous studies have shown that amaranth storage proteins possess encrypted sequences that, once released, exhibit different physiological activities. One of the most studied is antihypertensive activity. This review summarizes the progress made over the last years (2008-2020) related to this topic. Studies related to inhibition of different enzymes of the Renin-Angiotensin-Aldosterone system, in particular Angiotensin Converting Enzyme (ACE) and Renin, as well as those referring to potential modulation mechanisms of tissue or local Renin-Angiotensin-Aldosterone system, are analyzed, including in silico, in vitro, in vivo, and ex vivo assays. Furthermore, the potential use of these bioactive peptides or products containing them, in the elaboration of functional food matrices is discussed. Finally, the most relevant conclusions and future requirements in research and development of food products are presented.

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Determination of Antihypertensive Small Peptides, Val-Tyr and Ile-Val-Tyr, by Fluorometric High-Performance Liquid Chromatography Combined with a Double Heart-Cut Column-Switching Technique

Cited by 12 publications

References 15 publications

Benzotropolone moiety in theaflavins is responsible for inhibiting peptide-transport and activating AMP-activated protein kinase in Caco-2 cells

Benzotropolone moiety in theaflavins is responsible for inhibiting peptide-transport and activating AMP-activated protein kinase in Caco-2 cells

Application of 13C stable isotope labeling liquid chromatography–multiple reaction monitoring–tandem mass spectrometry method for determining intact absorption of bioactive dipeptides in rats

Amaranth as a Source of Antihypertensive Peptides

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