Methods, kits and devices for measuring extracellular pyridine nucleotide

Method for determining a concentration of oxidized extracellular pyridine nucleotide (ePNox) in a body fluid of a subject, comprising:

• a) providing the body fluid comprising the ePNox and reduced extracellular pyridine nucleotide (ePNred) and subjecting the body fluid to an extraction, wherein the extraction removes the ePNred from the body fluid to result in an extracted body fluid comprising ePNox and none or negligible amounts of ePNred,
• b) providing more than one pyridine nucleotide standard (PN standard) comprising aliquots of a standard matrix of the body fluid mixed with a known concentration of a pyridine nucleotide which are each subjected or have been subjected to an extraction, wherein the extraction removes or removed the ePNred from the PN standard to result in an extracted PN standard comprising ePNox and none or negligible amounts of ePNred,
• c) providing an oxidoreductase, a substrate of the oxidoreductase and at least one primary and at least one secondary redox indicator dye to the extracted body fluid of a) and each of the extracted PN standards of b) and subjecting the extracted body fluid and the extracted PN standards, respectively of a) and b) to a cycling process, wherein the cycling process comprises one or more cycles of:
• the oxidoreductase reducing the ePNox in the extracted body fluid of a) with a relative reaction velocity (VR) and in each of the extracted PN standards of b) to produce ePNred, wherein an electron is donated to the at least one primary redox indicator dye and subsequently to the at least one secondary redox indicator dye having an absorbance resulting in a change of the absorbance of the at least one secondary redox indicator dye,
• d) subsequently measuring the change in absorbance of the at least one secondary redox indicator dye in the extracted body fluid in which the ePNox was reduced to determine the VR in the extracted body fluid and measuring the change in absorbance of the at least one secondary redox indicator dye in each of the extracted PN standards in which the ePNox was reduced to determine the VR in each of the extracted PN standards and determining the concentration of the ePNox in the body fluid of the subject provided in a) based on the VR measured in the extracted body fluid relative to the VR measured in each of the extracted PN standards.