Verification of reference intervals of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in plasma on tandem mass spectrometry

Aminokiselina metionin i metaboliti metioninskog ciklusa S-adenozilmetionin (SAM), S-adenozilhomocistein (SAH) te aminotiol homocistein (Hcy), dio su tri važna stanična procesa, metilacije, remetilacije i transsulfuracije, a u tim procesima sudjeluju i mnogi enzimi i kofaktori (vitamin B6 , vitamin B12, folna kiselina). SAM je donor metilne skupine za brojne reakcije metilacije raznih makromolekula. Poremećaji procesa metilacije i remetilacije koji se očituju poremećenim koncentracijama SAM-a i SAH-a te poremećenim metilacijskim indeksom (MI, omjer SAM/SAH) mogu rezultirati brojnim patološkim procesima poput neurodegenerativnih bolesti, jetrenih i kardiovaskularnih bolesti, karcinoma i kongenitalnih abnormalnosti. Obzirom na uključenost SAM-a i SAH-a u brojne patološke procese, izvršena je verifikacija referentnih intervala naše populacije određivanjem koncentracija SAM-a i SAH-a te izračunavanjem metilacijskog indeksa u 50 odraslih volontera bez navedenih patoloških stanja. Prema preporukama struke, svaki je laboratorij dužan verificirati referentne intervale kako bi provjerio jesu li prikladni za populaciju bolesnika koju pokriva određeni laboratorij. Koncentracije SAM-a i SAH-a određivane su na UPLC-MS/MS (Shimadzu Nexera 8050) modificiranom metodom Gellekink i sur. Odgovarajuće pripremljeni uzorci i obilježeni interni standardi pročišćeni su pomoću kolona za ekstrakciju čvrsto-tekuće i injektirani u UPLC-MS/MS. Koncentracije SAM-a, SAH-a i izračunatog MI izražene su kao srednja vrijednost ± standardna devijacija kako bi se omogućila njihova usporedba s ostalim dostupnih literaturnim podacima. Srednja vrijednost ± SD za žene iznosila je: za SAM 60,3 ± 11,9 nmol/L, za SAH 29,4 ± 6,2 nmol/L, a za MI 2,1 ± 0,5. Srednja vrijednost ± SD za muškarce iznosila je: za SAM 76, 4 ± 29, 3 nmol/L, za SAH 39, 5 ± 15, 2 nmol/L, a za MI 2,0 ± 0,6. Zbog slične metode i pripreme uzoraka, naši rezultati su uglavnom usporedivi s onima koje je izvijestio Gellekink i sur., te ostalim studijama koje su također modificirale metodu prema Gellekink i sur. Opisana metoda može doprinijeti istraživanju bioloških odnosa između metilacije i brojnih patoloških stanja ispitivanjem referentnih vrijednosti na bolesnim ispitanicima. Zbog malog broja laboratorija koji se bave navedenim područjem i nedostupnosti literaturnih podataka, oni nisu primijenjivi u kliničkoj praksi.Amino acid methionine and methionine cycle metabolites S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and aminothiol homocysteine (Hcy) are part of three important cellular processes, methylation, remetylation and transsulfuration, in which are many enzymes and cofactors (vitamin B6, vitamin B12, folic acid) involved. SAM is a donor of methyl-groups for numerous methylation reactions of various macromolecules. Methylation and remetylation disorders that are manifested in disturbed SAM and SAH concentrations and disturbed methylation index (MI, SAM/SAH ratio) can result in numerous pathological processes such as neurodegenerative diseases, liver and cardiovascular diseases, cancer and congenital abnormalities. Given the involvement of SAM and SAH in numerous pathological processes, verification of reference intervals was done in our population by measuring SAM and SAH concentrations and calculation of methylation index in 50 adult volunteers without these pathological conditions. According to experts' recommendations, each laboratory is required to verify the reference intervals to test if it is suitable for the population of patients covered by a particular laboratory. Concentrations of SAM and SAH were determined on UPLC-MS/MS (Shimadzu Nexera 8050) using the modified method by Gellekink et al. Appropriately prepared samples and labeled internal standards were purified by using the columns for solid phase extraction and then injected in UPLC-MS/MS. Concentrations of SAM, SAH and calculated methylation indexes are represented as the mean ± standard deviation to allow comparison with other available literature data. For women mean ± SD for SAM is 60.3 ± 11.9 nmol/L, for SAH 29.4 ± 6.2 nmol/L and SAM/SAH ratio is 2.1 ± 0.5. For men mean ± SD for SAM is 76, 4 ± 29, 3 nmol/L, for SAH 39, 5 ± 15, 2 nmol/L and SAM/SAH ratio is 2.0 ± 0.6. Due to similar methods and sample preparation, our results are mostly comparable to those reported by Gellekink et al., and other studies which also modified the method according to Gellekink et al. The described method can contribute to the study of biological relationships between methylation and numerous pathological conditions by establishing the reference values in sick patients. Due to the small number of laboratories involved in this area and limited number of data publications, those publications are not applicable in clinical practice