Carnosine (CAR) and structurally related anserine (ANS) and homocarnosine (HCAR) are histidine-containing dipeptides widely distributed in vertebrate organisms. Although their biological role still remains unknown, it has been proposed that they act as highly effective quenchers of alfa,beta-unsaturated aldehydes such as 4-hydroxy-2-nonenal, nonenal, and acrolein. We recently proposed the reaction mechanism and found that the Michael adduct is the main reaction product (1-3). The aim of this work was to develop and validate a simple, sensitive and selective LC-MS/MS method to quantitate both histidine-dipeptides (HD) and the corresponding HNE-Michael adducts (HD-HNE) in biological matrices (rat tissues). Tissue homogenates (1:3 in PBS) from male Wistar rats were spiked with Tyr-His as internal standard (100 microM, final concentration) and deproteinized with 0.7 mM HClO4 (1:1 v/v). The supernatant was then separated on a Phenomenex Sinergy polar-RP column with a mobile phase consisting of water-acetonitrile-heptafluorobutyric acid (9:1:0.01 v/v/v) (phase A) and acetonitrile (phase B) at a flow rate of 0.2 ml min−1 and with a gradient elution. Detection was performed on an ion trap mass spectrometer equipped with an ESI interface operating in positive ionization mode. The acquisitions were performed in MRM mode using the following precursor > product ion combinations: Tyr-His (IS): m/z 319->301; CAR: m/z 227->210; ANS m/z 241->170,197,224; HCAR: m/z 241->156; carnosine-HNE Michael adduct (CHMA) m/z 383->366; anserine-HNE Michael adduct (AHMA) m/z 397->197, 241, 326, 353, 380. For HD analytes, the method was validated over the concentration range 15-1,000 nmoles g-1 (wet tissue) and the LOQ and LOD were 12.5 and 4.2 pmoles injected, respectively. The intra- and inter-day precisions (CV%) were < 10% (< 17.47% at the LOQ); intra- and inter-assay accuracy (RE%) were within ± 10% for all the concentrations. For HD-HNE analytes, the method was validated over the concentration range 10-100 nmoles g-1 and the LOQ and LOD were 8.1 and 3.0 pmoles injected, respectively. The mapping profile in rat tissue gave the following results: the highest concentrations of CAR and ANS were found in skeletal muscles (soleus, gastrocnemius, tibialis), ranging from 2.606 to 5.280 and 5.415-7.861 µmoles g-1 wet tissue respectively, followed by heart (67.03 and 64.50 nmoles g-1), cerebellum (48.41 and 57.48 nmoles g-1) and brain (27.35 nmoles g-1 CAR; ANS under LOQ). HCAR was found only in brain (51.14 nmoles g-1) and cerebellum (77.64 nmoles g-1). HD-HNE were under the LOD in all the tissue samples analysed. The present method will be used to profile HD and HD-HNE levels in several physiological (aging, endurance exercise) and pathological (inflammation, ischemia-reperfusion damage) conditions in order to reach the following goals: 1) to gain a deeper insight into the biological role of HD as detoxyfing agents of cytotoxic unsaturated aldehydes and to correlate the HD content to the carbonylation damage; 2) to find out whether HD-HNE can be used as specific, reliable and unequivocal markers of oxidative stress.

Determination of histidine-dipeptides and the corresponding 4-hydroxy-2-nonenal Michael adducts by liquid chromatography/electrospray ionization tandem mass spectrometry in rat tissues / G. Aldini, M. Orioli, M. Carini, R. Maffei Facino - In: Atti del congresso: Meeting of the HNE-Club[s.l] : HNE club, 2004. - pp. 18-18 (( Intervento presentato al 2. convegno Meeting of the HNE-Club tenutosi a Berlino nel 2004.

Determination of histidine-dipeptides and the corresponding 4-hydroxy-2-nonenal Michael adducts by liquid chromatography/electrospray ionization tandem mass spectrometry in rat tissues

G. Aldini
Primo
;
M. Orioli
Secondo
;
M. Carini
Penultimo
;
R. Maffei Facino
Ultimo
2004

Abstract

Carnosine (CAR) and structurally related anserine (ANS) and homocarnosine (HCAR) are histidine-containing dipeptides widely distributed in vertebrate organisms. Although their biological role still remains unknown, it has been proposed that they act as highly effective quenchers of alfa,beta-unsaturated aldehydes such as 4-hydroxy-2-nonenal, nonenal, and acrolein. We recently proposed the reaction mechanism and found that the Michael adduct is the main reaction product (1-3). The aim of this work was to develop and validate a simple, sensitive and selective LC-MS/MS method to quantitate both histidine-dipeptides (HD) and the corresponding HNE-Michael adducts (HD-HNE) in biological matrices (rat tissues). Tissue homogenates (1:3 in PBS) from male Wistar rats were spiked with Tyr-His as internal standard (100 microM, final concentration) and deproteinized with 0.7 mM HClO4 (1:1 v/v). The supernatant was then separated on a Phenomenex Sinergy polar-RP column with a mobile phase consisting of water-acetonitrile-heptafluorobutyric acid (9:1:0.01 v/v/v) (phase A) and acetonitrile (phase B) at a flow rate of 0.2 ml min−1 and with a gradient elution. Detection was performed on an ion trap mass spectrometer equipped with an ESI interface operating in positive ionization mode. The acquisitions were performed in MRM mode using the following precursor > product ion combinations: Tyr-His (IS): m/z 319->301; CAR: m/z 227->210; ANS m/z 241->170,197,224; HCAR: m/z 241->156; carnosine-HNE Michael adduct (CHMA) m/z 383->366; anserine-HNE Michael adduct (AHMA) m/z 397->197, 241, 326, 353, 380. For HD analytes, the method was validated over the concentration range 15-1,000 nmoles g-1 (wet tissue) and the LOQ and LOD were 12.5 and 4.2 pmoles injected, respectively. The intra- and inter-day precisions (CV%) were < 10% (< 17.47% at the LOQ); intra- and inter-assay accuracy (RE%) were within ± 10% for all the concentrations. For HD-HNE analytes, the method was validated over the concentration range 10-100 nmoles g-1 and the LOQ and LOD were 8.1 and 3.0 pmoles injected, respectively. The mapping profile in rat tissue gave the following results: the highest concentrations of CAR and ANS were found in skeletal muscles (soleus, gastrocnemius, tibialis), ranging from 2.606 to 5.280 and 5.415-7.861 µmoles g-1 wet tissue respectively, followed by heart (67.03 and 64.50 nmoles g-1), cerebellum (48.41 and 57.48 nmoles g-1) and brain (27.35 nmoles g-1 CAR; ANS under LOQ). HCAR was found only in brain (51.14 nmoles g-1) and cerebellum (77.64 nmoles g-1). HD-HNE were under the LOD in all the tissue samples analysed. The present method will be used to profile HD and HD-HNE levels in several physiological (aging, endurance exercise) and pathological (inflammation, ischemia-reperfusion damage) conditions in order to reach the following goals: 1) to gain a deeper insight into the biological role of HD as detoxyfing agents of cytotoxic unsaturated aldehydes and to correlate the HD content to the carbonylation damage; 2) to find out whether HD-HNE can be used as specific, reliable and unequivocal markers of oxidative stress.
Settore CHIM/08 - Chimica Farmaceutica
2004
Book Part (author)
File in questo prodotto:
Non ci sono file associati a questo prodotto.
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/152169
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact