A Facile and Sensitive Method for Quantification of Cyclic Nucleotide Monophosphates in Mammalian Organs: Basal Levels of Eight cNMPs and Identification of 2',3'-cIMP

Abstract

A sensitive, versatile and economical method to extract and quantify cyclic nucleotide monophosphates (cNMPs) using LC-MS/MS, including both 3',5'-cNMPs and 2',3'-cNMPs, in mammalian tissues and cellular systems has been developed. Problems, such as matrix effects from complex biological samples, are addressed and have been optimized. This protocol allows for comparison of multiple cNMPs in the same system and was used to examine the relationship between tissue levels of cNMPs in a panel of rat organs. In addition, the study reports the first identification and quantification of 2',3'-cIMP. The developed method will allow for quantification of cNMPs levels in cells and tissues with varying disease states, which will provide insight into the role(s) and interplay of cNMP signalling pathways.

Renal 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase Is an Important Determinant of AKI Severity after Ischemia-Reperfusion

“…The production of 29,39-cAMP as well as other nucleoside 29,39-cyclic monophosphates by living tissues has been corroborated by multiple independent laboratories. [26][27][28][29][30][31] Our hypothesis that injury increases 29,39-cAMP production also has received confirmation. For example, brain trauma in mice increases brain interstitial levels of 29,39-cAMP, and 29,39-cAMP is elevated in cerebrospinal fluid of humans in the early hours after traumatic brain injury.…”

Adenosine Production by Biomaterial‐Supported Mesenchymal Stromal Cells Reduces the Innate Inflammatory Response in Myocardial Ischemia/Reperfusion Injury

“…LV tissue was then snap frozen in liquid nitrogen, and kept at −80°C until further processing. Preparation of myocardial tissue for LC/MS was performed as previously described with the following modifications 36, 37. Frozen tissue was homogenized in prechilled acetonitrile/methanol/water (1:2:2, vol/vol/vol).…”

“…Preparation of myocardial tissue for LC/MS was performed as previously described with the following modifications. 36,37 Frozen tissue was homogenized in prechilled acetonitrile/ methanol/water (1:2:2, vol/vol/vol). Crude lysate was then heated for 10 minutes at 60°C, cooled on ice for 10 minutes, and then centrifuged at 2350g for 90 minutes at 4°C.…”

Recent progress in the field of cIMP research

“…Specifically in rat kidney and rat liver, low levels of cIMP were detected using sensitive and specific mass spectrometry methods (Jia et al, 2014). In addition, several organs from zebrafish, i.e., eyes, heart and testes, contain cIMP (Dittmar et al, 2015).…”

“…The available evidence suggests that the occurrence and the effects of cIMP may be species-specific (Jia et al, 2014;Dittmar et al, 2015;Detremmerie et al, 2016). Thus, numerous species beyond rat, mouse, rat, and human should be studied.…”